Conformationally constrained 2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T): synthesis, structure, physical, and biochemical studies of aza-ENA-T-modified oligonucleotides.
Conformationally constrained 2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T): synthesis, structure, physical, and biochemical studies of aza-ENA-T-modified oligonucleotides.
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构象限制的 2-N,4-C-乙烯桥胸苷 (aza-ENA-T)：aza-ENA-T 修饰寡核苷酸的合成、结构、物理和生化研究。
DOI：
10.1021/ja0634977
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发表时间：
2006
影响因子：
15
通讯作者：
J. Chattopadhyaya
中科院分区：
文献类型：
--
作者：
O. Varghese;J. Barman;W. Pathmasiri;O. Plashkevych;D. Honcharenko;J. Chattopadhyaya
2 -deoxy-2 - n, 4“-C-ethylene-bridged胸苷(aza-ENA-T)使用一个密钥环化一步合成涉及2》-ara-trifluoromethylsufonyl-4 ' -cyanomethylene 11给一对3 ',5 ' -bis-OBn-protected diastereomerically纯aza-ENA-Ts(12和12 b)融合piperidino骨架的椅式构象,而pentofuranosyl一半是锁定在North-type构象(7度< P < 27度,44度< mφ< 52度)。发现两种非对映纯aza-ENA-Ts的手性来源于内环手性2'-氮，其12b具有轴向N-H， 12a具有平伏N-H。后者在热力学上是优先的，而前者在动力学上是优先的，Ea = 25.4 kcal mol-1，这是迄今为止在双环胺中观察到的最高的N-H锥体反转势垒。采用5'-O-DMTr-aza-ENA-T-3'-磷酸酰胺进行固相合成，得到4种不同的单修饰15-mer反义寡核苷酸（AONs）。他们的AON/RNA双链显示，根据AON中的修饰位点的不同，每次修饰的温度升高2.5-4摄氏度。aza- ena - t修饰的AON/RNA异双工的RNase H1的相对切割速率与天然的AON/RNA非常相似，但修饰的AON/RNA的RNA切割位点却非常不同。与未修饰的AON（在4小时内完全降解）相比，aza-ENA-T修饰也使AON在血清中非常耐3'降解（在48小时内稳定）。因此，与天然修饰相比，AON中的aza-ENA-T修饰满足了三个重要的反义标准：(i)提高了RNA靶亲和力，（ii）相当的RNase H切割率，（iii）更高的血清稳定性。
The 2'-deoxy-2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T) 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) with the fused piperidino skeleton in the chair conformation, whereas the pentofuranosyl moiety is locked in the North-type conformation (7 degrees < P < 27 degrees, 44 degrees < phi m < 52 degrees). 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 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 thus 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 (AONs). Their AON/RNA duplexes showed a Tm increase of 2.5-4 degrees 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.