Exploring excited-state proton transfer reactions in isothiazologuanosine, an isofunctional fluorescent analogue of guanosine

Abstract

To fully exploit the potential of isothiazologuanosine (^tzG), an isomorphic and isofunctional fluorescent analogue of guanosine, as a probe for DNA and RNA, we characterized its photophysics and in particular its excited-state reactions over a wide pH range (–0.6 to 12) and time scale (100 fs–100 ns) by combining transient absorption and time-correlated single photon counting measurements with quantum mechanical calculations. At acidic pH, the dominant ground-state species ^tzG-H1–H3⁺, where the N atoms in positions 1 and 3 are protonated, rapidly converts to the more stable tautomer ^tzG-H1–H7⁺ in its excited state. The latter then deprotonates to form the ^tzG-H1 neutral species with an excited-state pK_a* value that differs by three pH units from the ground-state pK_a value. The rate constants governing the excited-state reactions and the fluorescence lifetime of each species were all determined. With the exception of intramolecular and solvent relaxations, no excited-state reactions in the femtosecond to nanosecond time scale were, however, observed between the dominant ^tzG-H1 and ^tzG-H3 tautomers in equilibrium at neutral pH or for ^tzG-H1 deprotonation at high pH. Because of the distinct spectra, fluorescence quantum yields and lifetimes of its different protonated and deprotonated forms, ^tzG is highly responsive over a wide range of acidic (0–5) and basic pH values (8–10). The mechanisms revealed herein will be instrumental for ^tzG-labelled oligonucleotides in order to interpret proton transfer reactions as well as interactions with specific protein domains, which, due to local electrostatic changes and water exclusion effects, may shift the pK_a values of ^tzG to a more physiologically relevant range.