Figure shows the molar absorption and fluorescence emission spectra of 5mCyd and Cyd in phosphate buffer, pH 7.4. At this pH, both molecules are neutral tautomers with pKa values of 4.3 and 4.2 for 5mCyd and Cyd, respectively. Upon methylation at the C5-position, a red shift of approximately 7 nm is observed, thereby allowing for more absorption into the UVB region of the electromagnetic spectrum reaching the Earth's surface. The absorption spectrum of each molecule shows three band maxima at ca. 278, 239, and 213 nm for 5mCyd and at 271, 232, and < 200 nm for Cyd. This red shift is supported by previous steady-state experiments and quantum chemical calculations in water performed at the MS-CASPT2/MM level of theory. Molar absorption coefficients of 7,942  62 and 9,337  30 were obtained for 5mCyd and Cyd at 278 and 271 nm, respectively. A red shift is also observed in the fluorescence emission spectrum of 5mCyd compared to that of Cyd, with maxima at ca. 345 nm and 330 nm, respectively, following excitation at 255 nm (Figure ). Using thymidine as a standard, a fluorescence quantum yield of 5.0 10 was estimated for 5mCyd, which is about 5 larger than the quantum yield for Cyd of 1.0 10 . These fluorescence quantum yields and spectral shifts are in good agreement with those previously reported by Sharonov et al. and Martínez-Fernández et al. for the 2'-deoxyribose analogs (5mdCyd and dCyd). The excitation spectra taken at the emission maxima align well with the lowest energy absorption band of 5mCyd and Cyd, respectively, indicating that the emission is an intrinsic property of these nucleosides (Figure ). Using the steady-state photophysical data and the Strickler-Berg equation, fluorescence lifetimes of 4.0 and 0.6 ps were estimated for 5mCyd and Cyd, respectively (see SI for details). These Strickler-Berg fluorescence lifetimes are in relatively good agreement with the experimentally obtained fluorescence lifetimes of 4.3 to 6.1 for 5mdCyd and 0.720 ps for Cyd. To investigate the electronic relaxation pathways of 5mCyd upon excitation at 267 nm, femtosecond broadband transient absorption spectroscopy was performed in back-to-back experiments with the Cyd. As described in detail in the SI, the transient absorption data were corrected for the hydrated electron signal that originates from the two-photon absorption of the water solvent using the method developed by Kohler and coworkers. Importantly, we also collected transient data exciting at 267 nm with low photon density conditions to avoid the formation of hydrated electrons from the solvent, as previously described. The latter results are shown in Figures . The transient absorption signals under both conditions are very similar from ca. 430 to 700 nm once the transient spectra under higher photon density conditions are corrected for the hydrated electron signal. However, as explained elsewhere and in the SI, the transient spectra in Figure is overcorrected from ca. 320 to 450 nm due to an additional coherent signal from the solvent within the cross correlation of the pump and probe beams in this spectral region (see Figure ). Following excitation of 5mCyd at 267 nm, a broad transient species is observed with maxima below 320 and 550 nm and a negative amplitude ΔA signal that grows in at ca. 350 nm (Figure ). This negative amplitude ΔA signal corresponds very well with the steady-state fluorescence emission and therefore, is assigned to stimulated emission. During the time delays between 0.15 and 0.99 ps, a decrease and blue shift in amplitude in the UV region is observed with a maximum at ca. 360 nm, while a small red shift is observed in the visible region (Figure ). Finally, as shown in Figure , a uniform decay across the entire probe region is observed between 0.99 and 31 ps. Similarly, for Cyd, a broad transient species is observed following excitation with maxima below 320 and at ca. 400 and 580 nm (Figure ). Between time delays of 0.13 and 3.2 ps, a decrease in the amplitude of the band at 580 nm is observed, while the band around 400 nm barely changes in amplitude (Figure ). From 3.2 to ca. 90 ps, the absorption bands with maxima below 320 and at ca. 400 nm fully decay and no other transient signal is observed at 2.7 ns (Figure ).