Methylated cytosine is proved to have an important role as an epigenetic signal in gene regulation and is often referred to "the fifth base of DNA". A comprehensive understanding of the electronic excited state relaxation in cytosine and its methylated derivatives is crucial for revealing UV-induced photodamage to the biological genome. Because of the existence of multiple closely lying "bright" and "dark" excited states, the decay pathways in these DNA nucleosides are the most complex and the least understood so far. In this study, femtosecond transient absorption with different excitation wavelengths (240-296 nm) was used to study the relaxation of excited electronic states of 5-methylcytosine (5mC) and 2'-deoxy-5-methylcytidine (5mdCyd) in phosphate buffered aqueous solution and in acetonitrile solution. Two distinct nonradiative decay channels were directly observed. The first one is a several picosecond internal conversion channel that involves two bright ππ* states (ππ* and ππ*) when ππ* state is initially populated. The second channel contains the lower energy ππ* state and a so far experimental unidentified long-lived state which exhibits a several nanosecond lifetime. The long-lived state can only be accessed by the initially excited ππ* state. Inspired by this new discovery in 5mC and 5mdCyd, we revisited the decay of excited state of 2'-deoxycytidine (dCyd), revealing very similar decay pathways. Additionally, a well-known dark nπ* state (carbonyl lone pair) with ∼30 ps lifetime is present in both decay channels in dCyd. With our detailed experimental results, we successfully reconcile the long history debate of cytosine excited state relaxation mechanism by pointing out that the reason for the complex dynamics under traditional 266 nm excitation is mixed signals from the above-mentioned two distinct decay pathways. Our findings lead to a dramatically different and new picture of electronic energy relaxation in 5mdCyd/dCyd and could help to understand photostability as well as UV-induced photodamage of these nucleotides and related DNAs.

DNA methylation and demethylation are the key steps in epigenetics. Emerging studies have demonstrated that these two processes play crucial roles in mammalian development and pathogenesis. Epigenetic modified cytosine and its further oxidative products, including 5-methylcytosine, 5-hydroxymethylcytosine, 5-formylcytosine, and 5carboxylcytosine, are called the "new four bases of DNA". The appearance of such new epigenetic bases can complicate DNA photodamage and repairing mechanism because they could have drastically different excited-state dynamics compared to canonical DNA nucleobases. In this study, excited-state dynamics of three demethylated nucleosides in buffer solution at physiological pH were investigated by femtosecond to microsecond time-resolved spectroscopy. Distinct excited-state dynamics are found in these demethylated nucleosides. For 2′-deoxy-5-formylcytidine (5fdCyd), direct observation of ultrafast intersystem crossing to the triple state with a 69% quantum yield is presented. Meanwhile, the triplet-state energy of 5fdCyd can transfer to the ambient molecular oxygen and generate destructive singlet oxygen. On the other hand, no such observation is seen in 2′-deoxy-5-hydroxymethylcytidine (5hmdCyd) and 2′-deoxy-5-carboxycytidine (5cadCyd), and these two bases show ultrafast internal conversion similar to that in 5-methylcytidine and cytidine. These results indicate that 5fdCyd is an effective internal triplet photosensitizer in DNA, and it could act as a new hot spot in DNA photodamage.

2'-Deoxy-5-formylcytidine (5fdCyd), a naturally occurring nucleoside found in mammalian DNA and mitochondrial RNA, exhibits important epigenetic functionality in biological processes. Because it efficiently generates triplet excited states, it is an endogenous photosensitizer capable of damaging DNA, but the intersystem crossing (ISC) mechanism responsible for ultrafast triplet state generation is poorly understood. In this study, time-resolved mid-IR spectroscopy and quantum mechanical calculations reveal the distinct ultrafast ISC mechanisms of 5fdCyd in water versus acetonitrile. Our experiment indicates that in water, ISC to triplet states occurs within 1 ps after 285 nm excitation. PCM-TD-DFT computations suggest that this ultrafast ISC is mediated by a singlet state with significant cytosine-to-formyl charge-transfer (CT) character. In contrast, ISC in acetonitrile proceeds via a dark 1 nπ* state with a lifetime of~3 ps. CTinduced ISC is not favored in acetonitrile because reaching the minimum of the gateway CT state is hampered by intramolecular hydrogen bonding, which enforces planarity between the aldehyde group and the aromatic group. Our study provides a comprehensive picture of the non-radiative decay of 5fdCyd in solution and new insights into the factors governing ISC in biomolecules. We propose that the intramolecular CT state observed here is a key to the excited-state dynamics of epigenetic nucleosides with modified exocyclic functional groups, paving the way to study their effects in DNA strands.

Abstract5‐Azacytosine (5‐AC) is one of the best interesting noncanonical nucleobases due to its functionalization and structural imitation of natural bases. 5‐AC can be used as the scaffold of two important chemotherapeutic medicines, 5‐azacytidine and 2′‐deoxy‐5‐azacytidine. Furthermore, increased sensitivity to UV leads to the photochemical effects of 5‐AC also attracted attention. Yet, no study has been reported to explore the effect of glycosyl groups on the photophysical and photochemical properties of 5‐AC, which can help to reveal the photostability of related actual clinic drugs. In this study, the excited‐state dynamics of 5‐azacytidine and 2′‐deoxy‐5‐azacytidine are studied by femtosecond transient absorption and quantum‐chemical calculations while revisiting that of 5‐AC with a wider probe spectral range. It is shown that glycosyl substitution on the N1 position leads to ultrafast excited‐state relaxation within several picoseconds in both nucleosides, which is distinct compared with the 17 ps lifetime seen in 5‐AC. It is proposed that these changes are due to altering the energy level of the dark nπ* state. Moreover, our results suggest that it should be cautioned to simply replace sugar groups with methyl groups when doing a theoretical calculation study on nucleobases and their derivatives.

We demonstrate that a single polycyclic π‐scaffold can undergo sequential multistep excited‐state structural evolution along the bent, planar, and twisted conformers, which coexist to produce intrinsic multiple fluorescence emissions in room‐temperature solution. By installing a methyl or trifluoromethyl group on the ortho‐site of N,N′‐diphenyl‐dihydrodibenzo[a,c]phenazine (DPAC), the enhanced steric effects change the fluorescence emission of DPAC from a dominant red band to well‐resolved triple bands. The ultra‐broadband triple emissions of ortho‐substituted DPACs range from ≈350 to ≈850 nm, which is unprecedented for small fluorophores with molecular weight of <500. Ultrafast spectroscopy and theoretical calculations clearly reveal that the above dramatic changes originate from the influence of steric hindrance on the shape of excited state potential energy surface (S1 PES). Compared to the steep S1 PES of parental DPAC, the introduction of ortho‐substituent is shown to make the path of structural evolution in S1 wider and flatter, so the ortho‐substituted derivatives exhibit slower structural transformations from bent to planar and then to twisted forms, yielding intrinsic triple emission. The results provide the proof of concept that the bent, planar, and twisted emissive states can coexist in the same S1 PES, which greatly expand the fundamental understanding of the excited‐state structural relaxation.