Excited‐State Dynamics of Thienoguanosine, an Isomorphic Highly Fluorescent Analogue of Guanosine

Abstract

Thienoguanosine (thG) is an isomorphic analogue of guanosine with promising potentialities as fluorescent DNA label. As a free probe in protic solvents, thG exists in two tautomeric forms, identified as the H1, being the only one observed in nonprotic solvents, and H3 keto–amino tautomers. We herein investigate the photophysics of thG in solvents of different polarity, from water to dioxane, by combining time‐resolved fluorescence with PCM/TD‐DFT and CASSCF calculations. Fluorescence lifetimes of 14.5–20.5 and 7–13 ns were observed for the H1 and H3 tautomers, respectively, in the tested solvents. In methanol and ethanol, an additional fluorescent decay lifetime (≈3 ns) at the blue emission side (λ≈430 nm) as well as a 0.5 ns component with negative amplitude at the red edge of the spectrum, typical of an excited‐state reaction, were observed. Our computational analysis explains the solvent effects observed on the tautomeric equilibrium. The main radiative and nonradiative deactivation routes have been mapped by PCM/TD‐DFT calculations in solution and CASSCF in the gas phase. The most easily accessible conical intersection, involving an out‐of plane motion of the sulfur atom in the five‐membered ring of thG, is separated by a sizeable energy barrier (≥0.4 eV) from the minimum of the spectroscopic state, which explains the large experimental fluorescence quantum yield.

Deciphering the pH-dependence of ground- and excited-state equilibria of thienoguanine

“…Supporting the results obtained for the th G nucleoside (21), only the H1 and H3 tautomers of th Gb are populated at neutral pH, the former being slightly more stable, in agreement with the experimental observations (Table S1 in SI). The computed vertical absorption energies are in good agreement with the experimental absorption spectra, but with a small overestimation (0.3 eV) of the transition energy of H3 (Table 1).…”

“…Geometry optimizations of the lowest energy bright excited state of H1 and H3 (identified as H1* and H3*) lead to stable minima of the Potential Energy Surface (PES), in line with the substantial experimental quantum yield of both tautomers. The computed emission energies and Stokes shift match fairly well with their experimental counterpart (Table 1) (21).…”

“…In a recent study, we demonstrated that th G exists as two ground-state tautomers with distinct absorption and emission spectra, and high quantum yield in aqueous buffer at neutral pH (14,21). Using quantum mechanical calculations, the two tautomers identified as the H1 and H3 keto-amino tautomers (Figure 1) were predicted to coexist both in the isolated nucleoside or when included in ODNs.…”

“…To model solvent effects, we resorted to a mixed discrete continuum model, where 6 water molecules were explicitly included in the calculations (Figure 1b) while bulk solvent effects are taken into account by the Polarizable Continuum Model (PCM) (28,29). This computational approach has already profitably been used to study th G, providing an accurate description of the tautomerization equilibria and the electronic spectra (21). We shall use the computed vertical absorption and emission energies to interpret the experimental spectra.…”

Dinâmica de estados excitados e propriedades espectroscópicas de derivados de DNA/RNA sintéticos e naturais em meio solvente

“…Dessa forma, acreditamos que essas moléculas também fluorescem em fase gasosa, apesar de não ter dados experimentais disponíveis. Uma recente investigação sobre a tienoguanina ( th I), uma análoga fluorescente das moléculas guanina e tz G, nos níveis TD-DFT e CASSCF, mostraram um caminho de desativação similar ao reportado por nós para a molécula tz G, com uma barreira energética ∼ 0.4 eV [225].…”