Ultrafast Excited-State Decay Mechanisms of 6-Thioguanine Followed by Sub-20 fs UV Transient Absorption Spectroscopy.

Understanding the primary steps following UV photoexcitation in sulphur-substituted DNA bases (thiobases) is fundamental for developing new phototherapeutic drugs. However, the investigation of the excited-state dynamics in sub-100 fs time scales has been elusive until now due to technical challenges. Here, we track the ultrafast decay mechanisms that lead to the electron trapping in the triplet manifold for 6-thioguanine in an aqueous solution, using broadband transient absorption spectroscopy with a sub-20 fs temporal resolution. We obtain experimental evidence of the fast internal conversion from the S2(ππ*) to the S1(nπ*) states, which takes place in about 80 fs and demonstrates that the S1(nπ*) state acts as a doorway to the triplet population in 522 fs. Our results are supported by MS-CASPT2 calculations, predicting a planar S2(ππ*) pseudo-minimum in agreement with the stimulated emission signal observed in the experiment.

Excited State Lifetimes of Sulfur-Substituted DNA and RNA Monomers Probed Using the Femtosecond Fluorescence Up-Conversion Technique.

Sulfur-substituted DNA and RNA nucleobase derivatives (a.k.a., thiobases) are an important family of biomolecules. They are used as prodrugs and as chemotherapeutic agents in medical settings, and as photocrosslinker molecules in structural-biology applications. Remarkably, excitation of thiobases with ultraviolet to near-visible light results in the population of long-lived and reactive triplet states on a time scale of hundreds of femtoseconds and with near-unity yields. This efficient nonradiative decay pathway explains the vanishingly small fluorescence yields reported for the thiobases and the scarcity of fluorescence lifetimes in the literature. In this study, we report fluorescence lifetimes for twelve thiobase derivatives, both in aqueous solution at physiological pH and in acetonitrile. Excitation is performed at 267 and 362 nm, while fluorescence emission is detected at 380, 425, 450, 525, or 532 nm. All the investigated thiobases reveal fluorescence lifetimes that decay in a few hundreds of femtoseconds and with magnitudes that depend and are sensitive to the position and degree of sulfur-atom substitution and on the solvent environment. Interestingly, however, three thiopyrimidine derivatives (i.e., 2-thiocytidine, 2-thiouridine, and 4-thiothymidine) also exhibit a small amplitude fluorescence component of a few picoseconds in aqueous solution. Furthermore, the N-glycosylation of thiobases to form DNA or RNA nucleoside analogues is demonstrated as affecting their fluorescence lifetimes. In aqueous solution, the fluorescence decay signals exciting at 267 nm are equal or slower than those collected exciting at 362 nm. In acetonitrile, however, the fluorescence decay signals recorded upon 267 nm excitation are, in all cases, faster than those measured exciting at 362 nm. A comparison to the literature values show that, while both the DNA and RNA nucleobase and thiobase derivatives exhibit sub-picosecond fluorescence lifetimes, the 1ππ* excited-state population in the nucleobase monomers primarily decay back to the ground state, whereas it predominantly populates long-lived and reactive triplet states in thiobase monomers.

A Unified Experimental/Theoretical Description of the Ultrafast Photophysics of Single and Double Thionated Uracils.

Photoinduced processes in thiouracil derivatives have lately attracted considerable attention due to their suitability for innovative biological and pharmacological applications. Here, sub-20 fs broadband transient absorption spectroscopy in the near-UV are combined with CASPT2/MM decay path calculations to unravel the excited-state decay channels of water solvated 2-thio and 2,4-dithiouracil. These molecules feature linear absorption spectra with overlapping ππ* bands, leading to parallel decay routes which we systematically track for the first time. The results reveal that different processes lead to the triplet states population, both directly from the ππ* absorbing state and via the intermediate nπ* dark state. Moreover, the 2,4-dithiouracil decay pathways is shown to be strongly correlated either to those of 2- or 4-thiouracil, depending on the sulfur atom on which the electronic transition localizes.

Radiation Induced One-Electron Oxidation of 2-Thiouracil in Aqueous Solutions.

Oxidative damage to 2-thiouracil (2-TU) by hydroxyl (•OH) and azide (●N3) radicals produces various primary reactive intermediates. Their optical absorption spectra and kinetic characteristics were studied by pulse radiolysis with UV-vis spectrophotometric and conductivity detection and by time-dependent density functional theory (TD-DFT) method. The transient absorption spectra recorded in the reactions of •OH with 2-TU depend on the concentration of 2-TU, however, only slightly on pH. At low concentrations, they are characterized by a broad absorption band with a weakly pronounced maxima located at λ = 325, 340 and 385 nm, whereas for high concentrations, they are dominated by an absorption band with λmax ≈ 425 nm. Based on calculations using TD-DFT method, the transient absorption spectra at low concentration of 2-TU were assigned to the ●OH-adducts to the double bond at C5 and C6 carbon atoms (3●, 4●) and 2c-3e bonded ●OH adduct to sulfur atom (1…●OH) and at high concentration of 2-TU also to the dimeric 2c-3e S-S-bonded radical in neutral form (2●). The dimeric radical (2●) is formed in the reaction of thiyl-type radical (6●) with 2-TU and both radicals are in an equilibrium with Keq = 4.2 × 103 M-1. Similar equilibrium (with Keq = 4.3 × 103 M-1) was found for pH above the pKa of 2-TU which involves admittedly the same radical (6●) but with the dimeric 2c-3e S-S bonded radical in anionic form (2●-). In turn, ●N3-induced oxidation of 2-TU occurs via radical cation with maximum spin location on the sulfur atom which subsequently undergoes deprotonation at N1 atom leading again to thiyl-type radical (6●). This radical is a direct precursor of dimeric radical (2●).

Dithionated Nucleobases as Effective Photodynamic Agents against Human Epidermoid Carcinoma Cells.

Sulfur-substituted nucleobases (i.e., thiobases) are a prospective class of compounds for clinical and cosmetic topical phototherapies. Recent investigations of several thiobases have revealed the ultrafast and efficient population of reactive triplet states upon ultraviolet-A (UVA) irradiation and the subsequent generation of singlet oxygen in high yield. In this contribution, we examine the photosensitizing activities of three of the most promising thiobase derivatives discovered to date: 2,4-dithiothymine, 2,4-dithiouracil, and 2,6-dithiopurine. These derivatives are shown to decrease the proliferation of human epidermoid carcinoma cells by up to 63 % in vitro, only upon activation with a low dose of UVA radiation (5 J cm-2 ). The generation of reactive oxygen species plays a minor role in the mode of action, suggesting these dithiobases may be effective within oxygen-deficient environments. Importantly, the photosensitized activity correlates with the magnitude of the triplet lifetime, which should guide the molecular design of next-generation photodynamic agents.