Photophysical characterization of isothiazologuanosine, a unique isomorphic and isofunctional fluorescent analogue of guanosine

Application of fluorescence techniques to investigate molecular interactions with nucleic acids is complicated by their poor emission, making the substitution of natural nucleobases by fluorescent nucleoside analogues (FNAs) a useful strategy. A breakthrough in fluorescent nucleoside analogues has been the development of thienoguanosine (thG) and isothiazologuanosine (tzG), two isosteric mimics of guanosine (G). Due to its N7 atom needed in Hoogsteen base pairs and enzyme recognition, tzG is also an isofunctional G surrogate. Herein, we integrated fluorescence spectroscopy measurements with quantum mechanical (QM) calculations to characterize the mechanisms underlying tzG photophysics in different solvents. In dioxane and ethyl acetate, tzG existed primarily as a H1 keto-amino tautomer with short fluorescence lifetime (r - 2 ns) and low quantum yield (O - 0.10). In buffer, the H1 tautomer (O = 0.36, r = 8.84 ns) coexisted with a weakly emissive H3 keto-amino tautomer. The two tautomers were also observed in methanol, but with a 30% decrease in O and r values for the major H1 tautomer. QM calculations suggested that the main non-radiative pathway of tzG-H1 involves NS bond loosening and is responsible for the more solvent-sensitive O and r values compared to thG. This pathway is much more efficient for tzG-H3, for which an additional pathway to a dark n pi* state and a large coupling with triplet states further explain its very low emission. This study lays the ground for rationally using tzG as a sensitive FNA.