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

Abstract

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.

Keywords: DNA and RNA derivatives; excited-state dynamics; fluorescence lifetimes; prodrugs; thiobases.

Scheme 1

Structures and common ring numbering of the thiobases.

Figure 1

Fluorescence up conversion signals of 6tGua and 6tGuo monomers at the emission wavelength of 380 nm upon excitation at 267 nm in PBS at pH 7.4.

Figure 2

Fluorescence up conversion data for (a) 2-thiopyrimidines, (b) 4-thiopyrimidines, (c) 2-thiouracil, 4-thiouracil, and 2,4-dithiouracil, and (d) 2,4-dithiopyrimidines in PBS at pH 7.4 at the emission wavelength of 380 nm upon excitation of the thiobase monomers at 267 nm.

Figure 3

Fluorescence up conversion data for (a) 2-thiouracil, 4-thiouracil, and 2,4-dithiouracil, (b) 4-thiopyrimidines, and (c) 2,4-dithiopyrimidines in acetonitrile at the emission wavelength of 380 nm upon excitation of the thiobase monomers at 267 nm.

Figure 4

Effect of solvent environment on the fluorescence decay of (a) 4tThd, (b) 4tUrd, and (c) 4tUra. Fluorescence decay lifetimes were measured in PBS at pH 7.4 and in acetonitrile (ACN), exciting at 267 nm and a detecting emission at 380 nm.