Conformationally constrained 2'-N,4'-C-ethylene-bridged thymidine (aza-ENA-T): synthesis, structure, physical, and biochemical studies of aza-ENA-T-modified oligonucleotides.
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.
Varghese
Oommen P
OP
Department of Bioorganic Chemistry, Box 581, Biomedical Center, Uppsala University, SE-75123 Uppsala, Sweden.
Barman
Jharna
J
Pathmasiri
Wimal
W
Plashkevych
Oleksandr
O
Honcharenko
Dmytro
D
Chattopadhyaya
Jyoti
J
D001483:Base Sequence; D001952:Bridged-Ring Compounds / Q000138:chemical synthesis / Q000737:chemistry; D004247:DNA / Q000097:blood / Q000737:chemistry; D004355:Drug Stability; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D019906:Nuclear Magnetic Resonance, Biomolecular; D009690:Nucleic Acid Conformation; D016376:Oligonucleotides, Antisense / Q000097:blood / Q000138:chemical synthesis / Q000737:chemistry*; D043264:Phosphodiesterase I / Q000737:chemistry / Q000378:metabolism; D013237:Stereoisomerism; D013816:Thermodynamics; D013936:Thymidine / Q000031:analogs & derivatives* / Q000097:blood / Q000138:chemical synthesis / Q000737:chemistry
D001952:Bridged-Ring Compounds; D016376:Oligonucleotides, Antisense; D004247:DNA; D043264:Phosphodiesterase I; C470790:phosphodiesterase I, snake venom; D013936:Thymidine