Synthesis and Structure of Novel Conformationally Constrained 1‘,2‘-Azetidine-Fused Bicyclic Pyrimidine Nucleosides:  Their Incorporation into Oligo-DNAs and Thermal Stability of the Heteroduplexes

Abstract

[structures: see text] The synthesis of novel 1',2'-aminomethylene bridged (6-aza-2-oxabicyclo[3.2.0]heptane) "azetidine" pyrimidine nucleosides and their transformations to the corresponding phosphoramidite building blocks (20, 39, and 42) for automated solid-phase oligonucleotide synthesis is reported. The novel bicyclonucleoside "azetidine" monomers were synthesized by two different strategies starting from the known sugar intermediate 6-O-benzyl-1,2:3,4-bis-O-isopropylidene-D-psicofuranose. Conformational analysis performed by molecular modeling (ab initio and MD simulations) and NMR showed that the azetidine-fused furanose sugar is locked in a North-East conformation with pseudorotational phase angle (P) in the range of 44.5-53.8 degrees and sugar puckering amplitude (phi(m)) of 29.3-32.6 degrees for the azetidine-modified T, U, C, and 5-Me-C nucleosides. Thermal denaturation studies of azetidine-modified oligo-DNA/RNA heteroduplexes show that the azetidine-fused nucleosides display improved binding affinities when compared to that of previously synthesized North-East sugar constrained oxetane fused analogues.

New methylene-bridged hexopyranosyl nucleoside modified oligonucleotides (BHNA): synthesis and biochemical studies

“…This is very similar to what we have observed earlier with other North-type conformationally constrained systems such as oxetane 15 azitidine 16,17 or aza-ENA 12 based AONs/RNA duplexes. This can be attributed to the local conformational change from DNA•RNA type to the RNA•RNA type conformation, which is not a substrate to RNase H. This local conformational change for a stretch of 5-6 nucleotides is however not detectable either by CD spectra or by the molecular model.…”

“…) or the carbocylic chain (carba-LNA 13 and carba-ENA 13,14 ), or by a covalent linkage between C1′ and C2′ (such as oxetane 15 or azetidine 16,17 types of constraints) result in to a conformationally-constrained North-type sugar. Oligonucleotides with these types of covalently constrained nucleosides can also potentially preorganize the single-stranded oligonucleotide in to a helical form, which, in turn, show much improved affinity toward complementary RNA (3-8ºC/modification, with the exception of C1′ and C2′ constrained systems) as well as to DNA 18 ;…”

Comparison of the RNase H Cleavage Kinetics and Blood Serum Stability of the North-Conformationally Constrained and 2‘-Alkoxy Modified Oligonucleotides

“…Electrostatic interaction between the positively charged modification and the enzyme can be responsible for the reduction in binding affinity. Therefore, the decrease of the enzyme binding affinity and a resulting loss in the effective enzyme activity for azetidine-T modified AON 5/RNA hybrid in comparison with the oxetane-T (26) counterpart AON 12/RNA can be attributed to the ability of the endocyclic 2′-amino function of azetidine-T to be protonated at the physiological pH (27) (pK a of 6.07). A similar reason may also be responsible in the case of aza-ENA-T modified AON 5, in comparison with its analogous ENA (46) modified AON, as the endocyclic aza-ENAnitrogen can also be protonated at the physiological pH (32), even more efficiently (pK a of 6.66) than the azetidine-T (pK a of 6.07).…”

“…Azetidine-T (27), aza-ENA-T (32) (Scheme S1 and Experimental Section in Supporting Information), 2′-O-Me-T (33), and 2′-O-MOE-T (34,35) modified nucleosides ( Figure 1) were synthesized following the previously reported procedures. Synthesis of modified oligonucleotides 1-11 and the complementary DNA (Table 1) was carried out using an Applied Biosystems 392 automated DNA/RNA synthesizer, as previously described (27). AONs were purified by 20% polyacrylamide 7 M urea denaturing gel electrophoresis (PAGE), extracted with 0.3 M NaOAc, and desalted with C18-reverse phase cartridges.…”

Chemical Modification of Oligonucleotides for Therapeutic, Bioanalytical and other Applications

“…This DNA modification (14) stabilises DNA-RNA heteroduplexes when compared to the iso-sequential oxetane-modified oligonucleotides (13), but still shows a destabilising effect (DT m /mod À1.0 to À5.5 8C) when compared to the native DNA-RNA heteroduplex. [19] Replacement of the 4'-oxygen of nucleosides by sulfur has been well established and subject to many studies. More recently Damha et al synthesised a DNA oligonucleotide con-taining a single 4'-selenonucleotide (15) insertion, which was found to stabilise duplex formation (DT m /mod +1.9 8C) for complementary RNA, but decrease stability (DT m /mod À1.9 8C) for complementary DNA despite the DNA-like conformation observed for 4'-Se-rT nucleosides.…”