Comparison of the base pairing properties of a series of nitroazole nucleobase analogs in the oligodeoxyribonucleotide sequence 5′-d(CGCXAATTYGCG)-3

The nucleoside analogs 1-(2′-deoxy-β-D-ribofuranosyl)-3-nitropyrrole (9), 1-(2′-deoxy-β-D-ribofuranosyl)-4-nitropyrazole (10), 1-(2′-deoxy-β-D-ribofuranosyl)-4-nitroimidazole (11) and 1-(2′-deoxy-β-D-ribofuranosyl)-5-nitroindole (21) were incorporated into the oligonucleotide 5′-d(CGCXAATTYGCG)-3′ in the fourth position from the 5′-end. Procedures for synthesis of two of the nitroazole nucleosides, 10 and 11, were developed for this study. Each of the nitroazoles was converted into a 3′-phosphoramidite for oligonucleotide synthesis by conventional automated protocols. Four oligonucleotides were synthesized for each modified nucleoside in order to obtain duplexes in which each of the four natural bases was placed opposite (position 9) the nitroazole. In order to assess the role of the nitro group on base stacking interaction, sequences were also synthesized in which the fourth base was 1-(2′-deoxy-β-D-ribofuranosyl)pyrazole. Corresponding sequences containing an abasic site, as well as sequences containing inosine, were synthesized for comparison. Thermal melting studies yielded Tm values and thermodynamic parameters. Each nucleoside analog displayed a unique pattern of base pairing preferences. The least discriminating analog was 3-nitropyrrole, for which Tm values differed by 5°C and ΔG25°C ranged from −6.1 to −6.5 kcal/mol. 5-Nitroindole gave duplexes with significantly higher thermal stability, with Tm values varying from 35.0 to 46.5°C and −ΔG25°C ranging from 7.7 to 8.5 kcal/mol. Deoxyinosine (22), a natural analog which has found extensive use as a universal nucleoside, is far less non-discriminating than any of the nitroazole derivatives. Tm values ranged from 35.4°C when paired with G to 62.3°C when paired with C. The significance of the nitro substituent was determined by comparison of the base pairing properties of a simple azole nucleoside, 1-(2′-deoxy-β-D-ribofuranosyl)pyrazole (12). The pyrazole-containing sequences melt at 10–20°C lower than the corresponding nitropyrazole-containing sequences. On average, the pyrazole-containing sequences were equivalent in stability (average ΔG = −4.8 kcal/mol) to the sequences containing an abasic site (average ΔG = −4.7 kcal/mol).