Synthesis and physico-chemical study of new fluorescent N-naphthimidazole phosphoramidate oligonucleotides

Abstract

This work describes a new class of backbone-modified oligonucleotides bearing an internucleotide PN-naphthimidazole phosphoramidate group, termed phosphoramidate polyaromatic azole oligonucleotides (PPAAOs). The modification is introduced via a Staudinger reaction with a synthesized 2-azido-naphthimidazole during automated solid-phase synthesis and is compatible with standard deprotection, purification and analytical procedures, enabling high-yield preparation of sequences with site-specific modifications. UV-Vis titration shows that a 5′-terminal PN-naphthimidazole group remains neutral over a wide pH range, whereas internal incorporation yields a pK_a of ∼6.8 and a switch from a neutral to an anionic state near physiological pH. Thermal denaturation experiments reveal decrease in the melting temperature of DNA/DNA and DNA/RNA duplexes (ΔT_m up to ∼4.8 °C and ∼11.9 °C, respectively), while circular dichroism spectroscopy indicates preservation of canonical B- and A-form of double helixes. The PN-naphthimidazole group imparts intrinsic fluorescence with intensity that depends on both pH and the position of the modification within the strand, and exhibits strong quenching when paired with apurinic/apyrimidinic (AP) sites, which is accompanied by stabilization of the damaged duplex. These combined thermodynamic, spectroscopic and structural features establish PN-naphthimidazole as a versatile polyaromatic phosphoramidate scaffold and highlight PPAAOs as promising environment-sensitive probes for DNA lesion detection, hybridization analysis and the design of nucleic acid based therapeutics.