Synthesis and enzymatic properties of aza-ENA modified nucleos(t)ides

The 2'-deoxy-2'-N,4'-C-ethylene bridged thymidine ''aza-ENA-T'' [PDF 366, 369] has been synthesized using a key cyclization step involving 2'-ara-trifluoromethylsufonyl-4'-cyanomethylene 11 to give a pair of 3',5'-bis-OBn protected diastereomerically pure aza-ENA-Ts (12a and 12b)

Scheme 1: Our strategy involving a ring-closure reaction to give 2',4'-conformationally constrained 2'-N,4'-C-ethylene bridged aza-ENA-T nucleoside D, where CN is first reduced to putative (B) followed by instantaneous cyclization (Structure C).

Since the 1',2'- North-East conformationally constrained oxetane and azetidine modified nucleoside (oxetane-T, -C, -A, -G [PDF 314, 315, 329, 332, 335 (C), 350 (G, A), 360], azetidine-T [PDF 363, 364]) have shown increased nuclease stability but deceased target affinity,  we have proceeded searching for compounds which would retain high nuclease resistance and possess high target affinity. That prompted us to design and synthesize the conformationally-constrained 2'-N,4'-C-ethylene bridged nucleic acid-thymidine (aza-ENA-T) which is an isostere of ENA-thymidine (2'-O,4'-C-ethylene bridged nucleic acid). This aza-ENA-T was incorporated into the AONs to explore their potential for effective gene-directed therapeutics and diagnostics. The aza-ENA based AONs may have three clear advantages over the corresponding ENA-containing counterparts in a similar manner as the 2'-amino-LNA modified AONs have over the LNA modified counterparts: First, the endocyclic amino functionality of the aza-ENA analog could be utilized as a well defined conjugation site and thereby we can control the hydrophilic, hydrophobic and steric requirements of a minor groove of the duplex. Second, the amine-derivatized AONs have displayed increased thermal affinities towards complementary RNA possibly because of the presence of positively charged moieties at physiological pH, and thus could influence partial neutralization of the negatively charged phosphates in the duplexes. Third, introduction of a fluorescence probe connected to this nitrogen moiety will enable us for real-time in vivo imaging of RNA and can therefore be used for specific detection of nucleic acids while maintaining their hybridization properties.

Text Box: Table 1. The aza-ENA-T modified AONs and the thermal Denaturation Studies of their duplexes with complementary RNA or DNA Targetsa.AONAON Sequences Synthesized Tm/CWith RNA?TmTm/C with DNA?Tm*MALDI-MS of AON 1 - 5: Found/calc [M+H]+13'-d(CTTCTTTTTTACTTC)-5'44454448.6/4448.723'-d(CTTCTTTTTTACTTC)48+444.5-0.54489.7/4491.1b33'-d(CTTCTTTTTTACTTC)46.5+2.542.5-2.54489.7/4490.743'-d(CTTCTTTTTTACTTC)47.5+3.542-34489.7/4490.753'-d(CTTCTTTTTTACTTC)48+442-34489.7/4490.8a Tm values measured as the maximum of the first derivative of the melting curve (A260 vs temperature) recorded in medium salt buffer (60 mM Tris-HCl at pH 7.5, 60 mM KCl, 0.8 mM MgCl2 and 2 mM DTT) with temperature range 20 to 70 oC using 1M concentrations of the two complementary strands; ?Tm = Tm relative to RNA compliment; ?Tm* = Tm relative to DNA compliment. T = aza-ENA-T monomer; b [M+2H]+.

The aza-ENA-T was synthesized and the structure was unequivocally proven by 1D and 2D NMR experiments and also by computational methods. The NMR and computational structural studies of aza-ENA-T showed that the piperidino moiety of the aza-ENA-T is indeed locked in the chair conformation (with the nitrogen lone-pair in the axial and N-H proton in the equatorial position), whereas the fused sugar is constrained to a North-type conformation similar to that of the 2'-O,4'-C-ethylene bridged ENA analog. Finally, the aza-ENA-T nucleotides have been incorporated in 15-mer AONs as single modification at four different sites to give four mono aza-ENA-T substituted AONs #2-5 (sequences shown in Table 1). These AON/RNA duplexes have shown an increase in the thermal stability by +2.5 to +4 °C per modification towards complementary RNA depending upon the substitution site. We also found that the relative rates of the RNase H1 promoted cleavage of the aza-ENA-T-modified AON/RNA heteroduplexes are comparable to that of the native counterpart, and, quite interestingly, the aza-ENA-T modifications also results in significant increase of AON/RNA resistance to 3'-exonucleases degradation in the blood serum compared to the native counterpart.

RNase H digestion studies of aza-ENA-T modified AON/RNA heteroduplexes

The antisense properties of aza-ENA-T modified oligonucleotides in duplex with the complementary RNA were compared with the native with Escherichia coli RNase H1 as a model system. Four different aza-ENA-T containing AON mixmers, each obtained by incorporating single aza-ENA-T modification at one of the four different positions (AONs 2 -5 in Table 1) of identical DNA sequence, when formed duplex with the complementary RNA, were found to be excellent substrate for RNase H1, but with varying RNA cleavage sites depending upon the site of modification on the AON strand (Figure 1). We have previously reported the RNase H1 digestion properties of oxetane modified AON/RNA hybrid duplexes in identical sequence. The RNA cleavage patterns of all aza-ENA-T modified AONs (AON 2-5 in Table 1) were found to be uniquely different from those of the isosequential oxetane modified AONs. AONs having oxetane modification showed cleavage footprint pattern of 5 nucleotide gap whereas AONs containing aza-ENA counterpart showed a gap of 6 and 7 nucleotide units. This shows that the local structures of all aza-ENA-T modified AONs/RNA duplexes are not the same. RNase H enzyme indeed can finely discriminate these local variations of the stereochemical properties of the microstructure brought about by various type and incorporation site of the North-type modification (aza-ENA-T versus oxetane modifications) in the AON.

Figure 1. (A) Autoradiograms of 20% denaturing PAGE, showing the cleavage kinetics of 5'-32P-labeled target RNA by E.coli RNase H1 in native AON 1/RNA (lane 5) and the aza-ENA-T modified AONs (2-5)/RNA hybrid duplexes (lane 1 to 4) after 2, 5, 15, 35 and 60 min of incubation. Conditions of cleavage reactions: RNA (0.8 µM) and AONs (4 µM) in buffer containing 20 mM Tris-HCl (pH 8.0), 20 mM KCl, 10 mM MgCl2 and 0.1 mM DTT at 21 °C; 0.08 U of RNase H. Total reaction volume 30 µL. (B) Kinetics of RNase H cleavage. Target RNA remaining is densitometrically evaluated and plotted as a function of time. Small inset shows the initial cleavage rates for clarity. (C) Pictorial representation of RNase H1 cleavage pattern of AONs 1-5/RNA hybrid duplexes. Vertical arrows show the RNase H cleavage sites, relative length of an arrow shows the relative extend of cleavage at that site and dotted arrows show the partial cleavage at the initial reaction time. Relative percentage cleavage is indicated above the arrow which is taken at 15 min time point from the gel shown above. (D) Quantitative evaluation of the gel picture of the remaining full length [32P]-RNA at 1 h as obtained by densitometer.

Stability of Aza-ENA-T/DNA chimeras in human serum

The stability of AON in cells towards various exo and endonucleases is warranted to fulfill the requirements for an ideal antisense agent. The stabilities of aza-ENA-T modified AONs were tested against human serum which mainly comprises of 3'-exonucleases (Figure 2). When compared to native counterpart which completely degraded after 9 h, AON 3, 4 and 5 (Table 1) were still remaining to certain extent (8, 15 and 20 % respectively). It is noteworthy that all modified AONs were cleaved by 3'-exonucleases in the blood serum at the phosphodiester which is one nucleotide before the aza-ENA-T modification site towards 3'-end, and the residual sequences were found to be stable in human serum for 48 h at 21°C .

Figure 2: Autoradiograms of 20% denaturing PAGE, showing the degradation pattern of 5'-32P-labeled AON 1 to 5 in human serum. Time points are taken after 0, 15, 30 min, 1, 2 and 9 h of incubation. The % of AON remaining after 9 h of incubation: 0% of AON 1, 0% of AON 2, 8% of AON 3, 15% of AON 4, 20% of AON 5.

This is a surprising result in view of the fact that identical AON sequences with North-constrained oxetane modification are cleaved at the phosphodiester immediately before the modification site under an identical condition.


Fused piperidino skeleton aza-ENA-T has been found  in the chair conformation, whereas the pentofuranosyl moiety is locked in the North-type conformation (7o < P < 27o, 44o < fm < 52o). The origin of the chirality of two diastereomerically pure aza-ENA-Ts was found to be due to the endocyclic chiral 2'-nitrogen, which has axial N-H in 12b and the equatorial N-H in 12a. The latter is thermodynamically preferred while the former is kinetically preferred with Ea = 25.4 kcal mol-1, which is so far the highest observed inversion barrier at pyramidal N-H in the bicyclic amines. The 5'-O-DMTr-aza-ENA-T-3'-phosphoramidite was employed for solid-phase synthesis to give four different singly-modified 15-mer antisense oligonucleotides (AON). Their AON/RNA duplexes showed a Tm increase of 2.5 to 4 °C per modification, depending upon the modification site in the AON. The relative rates of the RNase H1 cleavage of the aza-ENA-T-modified AON/RNA heteroduplexes were very comparable to that of the native counterpart, but the RNA cleavage sites of the modified AON/RNA were found to be very different. The aza-ENA-T modifications also made the AONs very resistant to 3'-degradation (stable over 48 h) in the blood serum compared to the unmodified AON (fully degraded in 4 h). Thus, the aza-ENA-T modification in the AON fulfilled three important antisense criteria, compared to the native: (i) improved RNA target affinity, (ii) comparable RNase H cleavage rate, and (iii) higher blood serum stability.

For further details on aza-ENA see the original papers [PDF 366, 369]