Phenyl-imidazolo-cytidine analogues: Structure-photophysical activity relationship and ability to detect single dna mismatch

To expand the arsenal of fluorescent cytidine analogues for the detection of genetic material, we synthesized para-substituted phenyl-imidazolo-cytidine (^PhImC) analogues 5a-g and established a relationship between their structure and fluorescence properties. These analogues were more emissive than cytidine (λ_em 398-420 nm, φ 0.009-0.687), and excellent correlation was found between φ of 5a-g and σ_p^- of the substituent on the phenyl-imidazolo moiety (R² = 0.94). Calculations suggested that the dominant tautomer of ^PhImC in methanol solution is identical to that of cytidine. DFT calculations of the stable tautomer of selected ^PhImC analogues suggested a relationship between the HOMO-LUMO gap and φ and explained the loss of fluorescence in the nitro analogue. Incorporation of the CF₃-^PhImdC analogue into a DNA probe resulted in 6-fold fluorescence quenching of the former. A 17-fold reduction of fluorescence was observed for the G-matched duplex vs ODN(CF₃-^PhImdC), while for A-mismatched duplex, only a 2-fold decrease was observed. Furthermore, since the quantum yield of ODN(CF₃-^PhImdC):ODN(G) was reduced 17-fold vs that of a single strand, whereas that of ODN(CF₃-^PhImdC):ORN(G) was reduced only 3.8-fold, ODN(CF₃-^PhImdC) appears to be a DNA-selective probe. We conclude that the ODN(CF₃-^PhImdC) probe, exhibiting emission sensitivity upon single nucleotide replacement, may be potentially useful for DNA single nucleotide polymorphism (SNP) typing.