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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. 

The substitution of an oxygen atom in an exocyclic carbonyl group of the nucleobases by a sulfur atom in a nucleic acid base generates a thiobase. This substitution causes a redshift in the absorption spectrum of the thiobase with respect to the canonical nucleobase, moving the strongly allowed absorption band from the UVC to the UVA region of the electromagnetic spectrum. Due to this redshift and the efficient population of the triplet state, 4-thiothymidine (4tThd) can be selectively excited without exciting canonical DNA, making it a powerful UVA photosensitizer. The synergistic toxicity of 4tThd and UVA radiation allows for the enhanced killing of skin cancer cells. As a result, 4tThd has been proposed for use in conjunction with UVA radiation as potential photodynamic therapy agent, due to its photochemical properties and to a diminished cytotoxicity. Studies of the monomer 4tThd have been performed to explore the prospective use of 4tThd in photochemotherapeutic application with reduced phototoxic side effects. One study of 4tThd in aqueous solution proposed the main kinetic mechanism to consist of intersystem crossing from the S2 state to the triplet manifold. Vertical excitation energies were calculated using the optimized ground state of 4tThd in water and vacuum. These were found to be in good agreement with the values previously reported. After studying the monomer, the next step is to understand what happens when 4tThd interacts with the DNA bases. Therefore, ground state optimizations and vertical excitation energies calculations were performed for a series of 4tThd-containing dinucleotides. These vertical excitation energy calculations predict the order electronic states and likely kinetic mechanisms when 4tThd is incorporated into DNA, which will greatly assist in the interpretation of planned time-resolved experiments.