Increased Photostability of the Integral mRNA Vaccine Component N₁‐Methylpseudouridine Compared to Uridine

Abstract

N 1 -Methylation of pseudouridine (m 1 ψ) replaces uridine (Urd) in several therapeutics, including the Moderna and BioNTech-Pfizer COVID-19 vaccines. Importantly, however, it is currently unknown if exposure to electromagnetic radiation can affect the chemical integrity and intrinsic stability of m 1 ψ. In this study, the photochemistry of m 1 ψ is compared to that of uridine by using photoirradiation at 267 nm, steady-state spectroscopy, and quantum-chemical calculations. Furthermore, femtosecond transient absorption measurements are collected to delineate the electronic relaxation mechanisms for both nucleosides under physiologically relevant conditions. It is shown that m 1 ψ exhibits a 12fold longer 1 ππ* decay lifetime than uridine and a 5-fold higher fluorescence yield. Notably, however, the experimental results also demonstrate that most of the excited state population in both molecules decays back to the ground state in an ultrafast time scale and that m 1 ψ is 6.7-fold more photostable than Urd following irradiation at 267 nm.

On the Photostability of Cyanuric Acid and Its Candidature as a Prebiotic Nucleobase

“…We note that this energy barrier is only 0.1 eV higher than the energy barrier found when LR-TD-DFT is used to find the (S 1 /S 0 ) CI. Thus, the qualitative agreement between the LR-TD-DFT and SF-TD-DFT results, together with the vast body of work that has reported analogous conical intersections for similar molecules [32][33][34][35][36][37], strengthen the idea of the prospective availability of S 1 /S 0 conical intersections and, as discussed above, the high photostability of this system. However, acknowledging the limitations of the methodology employed herein, we hope that these calculations will motivate theoretical groups that employ multireference methodologies to not only examine the availability of S 1 /S 0 conical intersections such as the one reported here, but to also investigate whether the higher lying S 5 , 6 (ππ*) states have access to conical intersections with the S 1 or the S 0 state out of the Franck-Condon region.…”

“…Furthermore, the molecular structure of CA resembles that of uracil, but with a carbon atom replacing the N5 atom and the addition of a carbonyl at the C6 position. Uracil monomers are photostable due to the availability of accessible conical intersections between the excited singlet states and the ground state [32][33][34][35][36][37], and they populate the triplet state with less than ca. 1% yield in an aqueous solution [38].…”

Photochemical Stability of 5-Methylcytidine Relative to Cytidine: Photophysical Insight for mRNA Therapeutic Applications

“…19 We remark, however, that all other transient absorption results reported in this Letter, and by Ma et al, 16 are consistent with those reported by Wang et al 19 To investigate the photoreactivity of 5mCyd relative to Cyd, low-intensity laser irradiation experiments were performed back-to-back exciting at 267 nm with a matched absorbance at the excitation wavelength (Figure 4), as has been reported recently for N1-methylpseudouridine and uridine. 33 Irradiation of both molecules results in a decrease in absorbance at the lowest energy band and at the higher-energy absorption band near 200 nm. A simultaneous increase in absorbance and red shift is observed for the absorption bands originally at 239 and 232 nm for 5mCyd and Cyd, respectively.…”

“…As described in detail in the Supporting Information, 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 co-workers . 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 S2–S6. The transient absorption signals under both conditions are very similar from ca.…”

“…To investigate the photoreactivity of 5mCyd relative to Cyd, low-intensity laser irradiation experiments were performed back-to-back exciting at 267 nm with a matched absorbance at the excitation wavelength (Figure ), as has been reported recently for N1-methylpseudouridine and uridine . Irradiation of both molecules results in a decrease in absorbance at the lowest energy band and at the higher-energy absorption band near 200 nm.…”