Ultrafast Excited-State Deactivation of 8-Hydroxy-2′-deoxyguanosine Studied by Femtosecond Fluorescence Spectroscopy and Quantum-Chemical Calculations

Abstract

The fluorescence properties of the 8-hydroxy-2'-deoxyguanosine (8-oxo-dG) in aqueous solution at pH 6.5 are studied by steady-state spectroscopy and femtosecond fluorescence up-conversion and compared with those of 2'-deoxyguanine (dG) and 2'-deoxyguanine monophosphate (dGMP). The steady-state fluorescence spectrum of 8-oxo-dG is composed of a broad band that peaks at 356 nm and extends over the entire visible spectral region, and its fluorescence quantum yield is twice that of dG/dGMP. After excitation at 267 nm, the initial fluorescence anisotropy at all wavelengths is lower than 0.1, giving evidence of an ultrafast internal conversion (<100 fs) between the two lowest excited ππ* states (Lb and La). The fluorescence decays of 8-oxo-dG are biexponential with an average lifetime of 0.7 ± 0.1 ps, which is about two times longer than that of dGMP. In contrast with dGMP, only a moderate dynamical shift (∼1400 vs 10,000 cm(-1)) of the fluorescence spectra of 8-oxo-dG is observed on the time scale of a few picoseconds without modification of the spectral shape. PCM/TD-DFT calculations, employing either the PBE0 or the M052X functionals, provide absorption spectra in good agreement with the experimental one and show that the deactivation path is similar to that proposed for dGMP, with a fast motion toward an energy plateau, where the purine ring keeps an almost planar geometry, followed by decay to S0, via out-of-the plane motion of amino substituent.

Excited State Relaxation of Neutral and Basic 8-Oxoguanine

“…Overall, our emission spectrum for neutral 8-oxo-dGuo is similar in appearance to the one in ref. 17 with maximum emission close to 350 nm. 8-oxo-dGuo − is considerably more fluorescent than the neutral form as seen in Figure 7a.…”

“…Although a more quantitative picture of the dynamics in solution should include explicit calculations along the paths, we believe the current results point to a small effect of solvation. 17 Whereas the authors of ref. 17 report a quantum yield of (2.3 ± 0.1) × 10 −4 for neutral 8-oxo-dGuo, we measure a somewhat higher value of (2.9 ± 0.4) × 10 −4 .…”

“…17 Whereas the authors of ref. 17 report a quantum yield of (2.3 ± 0.1) × 10 −4 for neutral 8-oxo-dGuo, we measure a somewhat higher value of (2.9 ± 0.4) × 10 −4 . Overall, our emission spectrum for neutral 8-oxo-dGuo is similar in appearance to the one in ref.…”

“…This assumption for 8-oxo-dGuo is supported by recent studies which show that indeed the base dominates the dynamics. 17 Furthermore, previous studies suggest that the lifetimes of ππ * states are not affected much by the addition of the sugar. 18 Recent work has shown differences between the excited state lifetime of the free base adenine and its nucleoside adenosine, 19 which have been explained using a proton-coupled electron transfer (PCET) deactivation mechanism involving the N 3 of the adenine residue and the hydrogen of the 5 ′ hydroxyl of the ribose group.…”

Second-order photon correlation measurement with picosecond resolution using frequency upconversion

“…On another note, frequency conversion using a short laser pulse can also provide high time resolution [6]. This technique has been used to characterize the fluorescence decay of a wide range of physical systems under pulsed excitation, with a resolution below a picosecond [7][8][9][10]. In these experiments, the emitted light is mixed in a nonlinear crystal with a short laser pulse (termed pump pulse) that is synchronized with the excitation laser.…”