SYNTHESIS OF CONFORMATIONALLY CONSTRAINED 2'-N,4'-C-ETHYLENE-BRIDGED ADENOSINE (aza-ENA-A)

The synthesis of conformationally constrained 2'-N,4'-C-ethylene-bridged adenosine (aza-ENA-A), in which the pentose-sugar is cis-fused with the piperidino skeleton at C2' and C4' centres of the sugar ring, is reported. The corresponding phosphoramidite building block will be used for incorporation into oligo-DNA and -RNA by solid phase synthesis to examine their nuclease stability as well as their application in blocking the translation of the target RNA using the antisense and siRNA approach. 2-Aza-6-oxabicyclo[3.2.1]octane skeleton is assembled through multi-step synthetic manipulation of appropriately protected D-arabinose based sugar precursor. The conversion of appropriate arabino precursor to ribo counterpart was achieved by direct nucleophilic displacement of “ara” positioned 2-(trifluoromethanesulfonyloxy) group in the sugar precursor 8. A high regioand enhanced stereoselectivity with preferential formation of β anomer in glycosylation reaction was achieved using Vorbrüggen conditions in the absence of any 2-participating group. Coupling step was performed using 1-O-acetyl-3,5-di-O-benzyl-(2-deoxy-2-azido)-4-C-(p-toluoyloxyethyl)-D-ribofur anose (10) as a glycosyl donor and persilylated N-benzoyladenine. Finally, the ring-closure giving the North-type conformationally constrained cis-fused bicyclic aza-ENA-A have been confirmed unambiguously by the long range H-C NMR correlation (HMBC), TOCSY, COSY and nOe experiments. † In memoriam – Professor Ivar Ugi UN CO RR EC TE D PR OO F INTRODUCTION Antisense oligonucleotides (AONs) incorporated with sugar-modified nucleosides have been successfully used as valuable tools to inhibit gene expression by utilizing various mechanisms of actions. Once bound to the target RNA, antisense agent either sterically blocks the synthesis of ribosomal proteins or induces RNase H mediated degradation of the target mRNA. For in vivo applications of the oligonucleotide-based approaches, appropriate chemical modifications are used to enhance target affinity, specificity, stability towards the endoand exonucleases, as well as to achieve tissue-specific delivery in order to improve the overall pharmacokinetic properties. 5, 8-11 Among these, the AONs modified with North-East conformationally (-1o < P < 34 o) constrained (LNA, ENA, oxetanes and their aza-analogues) 13-20 nucleotide blocks have unique abilities to dictate conformational pre-organization of the AON-RNA duplex to the rigid RNA-RNA type duplex which results in the modulation of the target affinity. We have previously reported synthesis of new 2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T) (2-aza-6-oxabicyclo[3.2.1]octane skeleton as sugar part in compound A in Figure 1), which has been subsequently incorporated into AONs and their antisense properties have been evaluated in vitro. The thermal stability of duplexes involving aza-ENA-T was determined toward the complementary RNA and DNA. Single modification was incorporated at a time at different positions of the 15nt AON sequence, 3'-d(CTTCTTTTTTACTTC)-5', to determine the sequence dependency of the target affinity toward complementary RNA and DNA. The results revealed that the single aza-ENA-T modification enhances the target affinity significantly with complementary RNA (∆Tm +2.5 to +4 °C) depending upon the site of the modification in the AON strand. This can be attributed to the site-dependency of the variable conformational pre-organization character imparted by the North-fused sugar moiety on the single stranded AON. The 2'-amino function of aza-ENA-T is almost 50% protonated at the physiological pH considering its pKa of 6.66 ± 0.03, and can have electrostatic interaction with the neighboring phosphate, which favors efficient duplex formation as observed in azetidine modified ODNs. On the other hand, with complementary DNA there was significant drop in duplex melting. This can be due to the 2',4'-ethylene-bridge which causes steric clash in the minor groove of the AON-DNA duplex. All of the aza-ENA-T modified AON-RNA hybrid duplexes have been found to be good substrates for the E. coli RNase H1 and comparable to that of the native counterpart. They also showed considerably stability in human blood serum (over 48 h) as well as toward snake venom phosphodiesterase compared to the native sequence. Thus the successful design of the conformationally constrained aza-ENA-T modified oligos with the North-fused sugar moiety and the protonable endocyclic-2'-aza function at physiological pH, as two key factors enhancing the stability of AON-RNA duplexes, prompted us to investigate the properties of aza-ENA-adenosine analogue (compound B in Figure 1). Compared to the earlier report of the thymine UN CO RR EC TE D PR OO F derivative of aza-ENA (aza-ENA-T), the synthesis required completely new and unique approach simply because the assistance by neighboring group participation of a pyrimidine is absent in the purine counterpart. Thus, it is noteworthy that synthesis of all azaor thio-2',4'-bridged nucleosides 16, 18-20 as well as aza-1',2'-cis-fused nucleoside analogues, which are so far reported in the literature, require an “ara” positioned 2'-hydroxyl group in the sugar, which is first converted into a leaving group for its subsequent displacement with various nucleophiles via an SN2 reaction. As stated above, this approach is particularly suitable for the synthesize of only pyrimidine analogues of 2'-deoxy-2'-N,4'-C-constrained nucleosides, because it is easy to form 2,2'-O-anhydro analogues by nucleophilic attack of C=O at the C2 centre of uracil or thymine. Recently, the synthesis of 2'-deoxy-2'-N,4'-nucleosides by transglycosylation reaction has also been reported; this approach has however severe limitation because of poor accessibility of the appropriate pyrimidine analogues.