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Abstract N1‐Methylation of pseudouridine (m1ψ) 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 m1ψ. In this study, the photochemistry of m1ψ 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 m1ψ exhibits a 12‐fold longer1ππ* 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 m1ψ is 6.7‐fold more photostable than Urd following irradiation at 267 nm.more » « less
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