1',2'-Azetidine modified nucleos(t)ides

The North-East type conformationally constrained nucleosides have been achieved also in the case of 1',2'-azetidine type of modification.
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  (Figure 1) for automated solid-phase oligonucleotide synthesis is reported [PDF 363, 364]. 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 44.5º to 53.8º and sugar puckering amplitude (fm) of 29.3º to 32.6º for the azetidine modified T, U, C, and 5Me-C nucleosides. Thermal denaturation studies of azetidine-modified oligo-DNA/RNA heteroduplexes show that the azetidine fused nucleosides [PDF 363, 364] display improved binding affinities when compared to that of previously synthesized North-East sugar constrained oxetane fused analogues [PDF 314, 315, 329, 332].
 

Azetidine-T
Azetidine-U
Azetidine-C
Azetidine-5-Me-C
Figure 1. Chemical structures of the azetidine modified pyrimidine nucleosides building blocks.

Fused azetidine derivatives of T, U and C phosphoramidites building blocks were synthesized from 6-O-benzyl-1,2:3,4-bis-O-isopropylidene-D-psicofuranose, and incorporated into 15mer AONs.  Thermal denaturation studies of azetidine-modified oligo-DNA/RNA heteroduplexes (Table 1) show that the azetidine fused nucleosides display improved binding affinities when compared to that of previously synthesized North-East sugar constrained oxetane fused analogues.
 

Table 1. Sequences Synthesized and Thermal Denaturation Studies towards Complementary RNA Targets and Comparison with Oxetane-Modified Sequences. DTmTm relative to native duplex; DTm* = Tm relative to oxetane modified duplex; “nd” = not determined.
AON
Sequence
DTm (C)
DTm
Tm*
1
3'-d(CTTCTTTTTTACTTC)-5'
44
   
2

2a

3'-d(CTT-azeCTTTTTTACTTC)
3'-d(CTT-oxeCTTTTTTACTTC)
38.5

38

-5.5

-6

+0.5
3

3a

3'-d(CTTCTT-azeTTTTACTTC)
3'-d(CTTCTT-oxeTTTTACTTC)
40

39

-4

-5

+1
4

4a

3'-d(CTTCTTTT-azeTTACTTC)
3'-d(CTTCTTTT-oxeTTACTTC)
40

40

-4

-4

0
5

5a

3'-d(CTTCTTTTTT-azeACTTC)
3'-d(CTTCTTTTTT-oxeACTTC)
40

39

-4

-5

+1.0
6
3'-d(CTU-azeCTTTTTTACTTC)
39.5
-4.5
nd
7
3'-d(CTTCTU-azeTTTTACTTC)
40.5
-3.5
nd
8
3'-d(CTTCTTTU-azeTTACTTC)
41
-3
nd
9
3'-d(CTTCTTTTTU-azeACTTC)
40
-4
nd
10

10a

3'-d(CTTC-azeTTTTTTACTTC)
3'-d(CTTC-oxeTTTTTTACTTC)
43.0

41.0

-1.0

-3

+2.0
11
3'-d(CTTCTTTTTTAC-azeTTC)
42
-2
nd

The molecular structures of these monomer units have been studied by means of high-field NMR and theoretical ab initio and MD simulations. Conformational analysis performed by molecular modeling showed that the azetidine-fused furanose sugar is locked in a North-East conformation with pseudorotational phase angle (P) in the range of 44.5º to 53.8º and sugar puckering amplitude (fm) of 29.3º to 32.6º for the azetidine modified T, U, C, and 5Me-C nucleosides. The combined experimental NMR data and theoretical simulations show that azetidine modification leads sugar moiety into locked North-East conformation with the puckering amplitudes and phase angles similar to those observed in the class of oxetane modified nucleosides. However, the relatively less electronegative nature of the nitrogen atom compared to the oxygen atom decreases the strength of restrains introduced by the azetidine moiety on the sugar conformation. And although it leads to very similar average sugar conformation, the dynamics of the sugar pucker in the azetidine modified nucleoside increases P fluctuation amplitude by almost 30%. Broader conformational subspace available for the azetidine modification can potentially be a decisive factor for the in vivo and in vitro applications making these oligos better substrate for a selected target.
 

For further details on  azetidine-T see the original papers [PDF 363, 364]