Excited State Decay Pathways of 2′-Deoxy-5-methylcytidine and Deoxycytidine Revisited in Solution: A Comprehensive Kinetic Study by Femtosecond Transient Absorption

Abstract

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.

Quantum dynamics of the ππ*/nπ* decay of the epigenetic nucleobase 1,5-dimethyl-cytosine in the gas phase

“…in water from ≤1 ps to 6∼9 ps. 11,12,14,15,18 A similar trend is found also for jet-cooled molecules in the gas phase. 16 This result has been related to the non-radiative decay path of the lowest energy bright transition, which is a HOMO→LUMO ππ * excitation for both for Cyt, 5Me-Cyt and 1,5diMe-Cyt, often used as a model for 5Me-dCyd.…”

“…5,6 In particular it is more prone than cytosine (Cyt), its parent base, to undergo photodimerization. [7][8][9][10] The photoactivated dynamics of 5Me-Cyt and 5-methyl(deoxy)cytidine (5Me-dCyd) nucleoside present in DNA has thus been rather extensively studied by experiments 9,[11][12][13][14][15][16] and calculations. 10,13,[16][17][18][19][20][21] Methyl substitution at position 5 has been shown to increase the excited state lifetime of Cyt by almost one order of magnitude, i.e.…”

“…16,18 It involves the so-called C5-C6 ethylenic Conical Intersection (CoI), characterized by the motion of the C5 and C6 substituent out from the molecular plane; the associated energy barrier, very small for Cyt, is increased by 5-methylation. 16,18 A methyl in position 5 also suppresses a deactivation channel, 14,15 with a characteristic time of ∼30 ps, identified for Cyt and (deoxy)Cytidine (dCyd) in water 14,15 and in chloroform, 22 and assigned to a dark nπ * transition. 18 Actually, different computa-tional models predict that, for both Cyt and 5Me-Cyt, two 'dark' nπ * states, involving the Lone Pair of N3 and O7, fall in the energy range of the two lowest energy ππ * transitions.…”

Triplet‐Induced Lesion Formation at CpT and TpC Sites in DNA

“…Additionally,t he photophysics of individual thymine and cytosine derivatives are still the topic of current research (e.g.,s ee refs. [26][27][28][29][30]), building the foundation for ad etailed understanding of photoreactions in the corresponding dipyrimidines.…”

pH Controlled Intersystem Crossing and Singlet Oxygen Generation of 8‐Azaadenine in Aqueous Solution

“…Excited‐state dynamics of 8‐AA were investigated using broadband femtosecond transient absorption spectroscopy. The experiment setup has been reported in the previous investigation . Briefly, a Ti:sapphire amplifier (Astrella, Coherent Inc.) generates a fundamental beam with the power output of 7 W. Almost 500 mW of the fundamental beam is adopted to generate a deep UV 266 nm pulse by third harmonic generation.…”