Excited State Decay Pathways of 2â²-Deoxy-5-methylcytidine and

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B: Biophysics; Physical Chemistry of Biological Systems and Biomolecules

Excited State Decay Pathways of 2’-deoxy-5-Methylcytidine and Deoxycytidine Revisited in Solution: A Comprehensive Kinetic Study by Femtosecond Transient Absorption Xueli Wang, Zhongneng Zhou, Yuankai Tang, Jinquan Chen, Dongping Zhong, and Jian-Hua Xu J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.8b00927 • Publication Date (Web): 25 Jun 2018 Downloaded from http://pubs.acs.org on June 26, 2018

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The Journal of Physical Chemistry

Excited State Decay Pathways of 2’-deoxy-5-Methylcytidine and Deoxycytidine Revisited in Solution: A Comprehensive Kinetic Study by Femtosecond Transient Absorption

Xueli Wang,1 Zhongneng Zhou,1 Yuankai Tang,1 Jinquan Chen,1,2* Dongping Zhong3 and Jianhua Xu1,2

1. State Key Laboratory of Precision Spectroscopy, East China Normal University, Shanghai, 200062 China 2. Collaborative Innovation Center of Extreme Optics, Shanxi University, Taiyuan, Shanxi 030006, China 3. Department of Physics, Department of Chemistry and Biochemistry, Programs of Biophysics, Chemical Physics, and Biochemistry, The Ohio State University, Columbus, OH 43210, United States

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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 most complex and 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 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*2 and ππ*1) when ππ*2 state is initial populated. The second channel contains the lower energy ππ*1 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*1 state. Inspired by this new discovery in 5mC and 5mdCyd, we revisited the decay of excited state of 2’-deoxycytidine (dCyd), revealing very similar distinct decay pathways. Additionally, a well-known dark nOπ* 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 2


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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.

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Introduction UV-induced photodamage to biological genome is initiated by excited electronic states formed in DNA through the absorption of UV photons. Excitation of DNA is highly efficient due to the strong ππ* transitions of the nucleobases. Typically, singlet excited states of nucleobase monomers decay in hundreds of femtoseconds to the electronic ground state due to nearly barrierless pathways leading from the Franck– Condon (FC) region to S1/S0 conical intersections (CIs). Because of the biological importance of these processes, a comprehensive understanding of the rapid nonradiative decay pathways which deactivate excited states of DNA nucleobases and polynucleotides has been the goal of many investigators in the past decade and a number of experimental as well as theoretical studies have been documented since year 2000.1-12 However, there are evidences showing the existence of longer lived electronic excitations in DNA. For example, charge transfer states with pico- to nanosecond lifetimes were observed in base pairs, single and double stranded DNAs due to base stacking and base pairing.

5, 11, 13-20

Meanwhile, longer lived “dark” electronic excited

states have also been detected in monomeric DNA bases. Pyrimidine base cytosine and its derivatives are of special interest because they exhibit the most complex and controversial excited state dynamics in solution. While earlier femtosecond transient absorption2, 21 and fluorescence up-conversion22 experiments suggest a ~1 ps lifetime for the lowest bright ππ* state, more recent experimental studies

23-28

indicate the

existence of a longer-lived dark nπ* state. On the other hand, a number of quantum 4


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mechanical calculations showed that there are two bright ππ* and two dark nπ* states with energies between 4.5 and 6.0 eV in cytosine9, 12, 29-37 and the debate about the preferential nonradiative decay pathways through different accessible conical intersections (CIs) between these four excited states and the ground state has never been fully disclosed. Furthermore, DNA methylation has an important role in mammalian development and the most common methylation is seen at the 5-position of cytosine. Thus, 5-methylcytosine is referred to “the fifth base of DNA” due to its importance in epigenetics.38-41 Methylation at the C5 position not only regulates gene expression but also alters the excited state dynamics of cytosine, leading to ~6-8 times longer excited state lifetime21, 26, 37, 42 and an increasing of cyclobutane-pyrimidine dimer (CPD) formation quantum yield.

43-44

The effect of C5 methylation on the

excited state relaxation dynamics of cytosine, especially those involving the dark nπ* state, is still elusive although it has been addressed by two independent studies recently. 26, 37 Ma et al. studied excited states dynamics of cytosine and its DNA and RNA nucleosides and nucleotides as well as the C5 methylated nucleosides in water and methanol by femtosecond transient absorption and fluorescence up-conversion.

26

They claimed that in cytosine and its N1-derivatives there are one sub-picosecond and another ~1 ps decay that account for the decay from ππ* state to ground state (S0) while an additional dark nπ* state exist. However, only one ππ* state decay was seen in C5 methylated cytosine. Recently, Martínez-Fernández and co-workers reported a quantum mechanical calculation study on deoxycytidine and 5-methyldeoxycytidine 5



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excited states relaxation mechanism.37 They assigned a tens of picoseconds decay component in deoxycytidine to a dark nOπ* state and proposed a low-lying nNπ* dark state which could also participate in the excited state dynamics. Furthermore, they pointed out that a second bright ππ* state in deoxycytidine could be populated by 267 nm excitation and this state is a “doorway” state for the nOπ* state. Meanwhile, C5-methylation could destabilize both nOπ* and nNπ* dark states and they are not involved in the decay dynamics at all. Unfortunately, there is no experimental evidence that fully support their calculations and the question about how nπ* dark states affect the excited states dynamics of cytosine and its C5 methylated derivatives remains unsolved. To the best of our knowledge, the excitation wavelength is always 266 nm in all previous experimental studies on excited state dynamics of cytosine and its C5 methylated derivatives in aqueous solution.2, 21-27, 45 This pump wavelength (Figure 1) is problematic because it leads to a non-negligible population of a second higher energy bright ππ* state37 which could bring complexity and obscure the truth of the excited state relaxation mechanism for cytosine. In this study, we conducted an excited

state

dynamics

study

on

5-methylcytosine

(5mC)

and

2’-deoxy-5-methylcytidine (5mdCyd) in phosphate buffer and acetonitrile solutions by using femtosecond broadband transient absorption with different excitation wavelengths (240-296 nm). By varying the excitation wavelength, we discovered two distinct decay pathways in 5mC and 5mdCyd when they populate different initial bright ππ* states. Moreover, an undocumented long-lived dark state was directly 6


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observed in 5mC and 5mdCyd for the first time. After these new results, we revisited the excited state dynamics of 2’-deoxycytidine (dCyd) in buffer and acetonitrile solutions and discovered similar decay mechanism. The results we obtained provide direct evidence for clarifying the disagreement and disclosing the long history debate on excited state dynamics of cytosine and its C5 methylated derivatives. This study also provides valuable fundamental for understanding the behavior of these bases in single and double strand DNAs.

Figure 1. UV/vis absorption spectra and structure of (a) 5mdCyd and (b) dCyd.

Experimental Methods 5-Methylcytosine (5mC) (95%), 2’-deoxycytidine (dCyd) (98%), acetonitrile (99.9%, super dry) were purchased from J&K chemical Ltd. (Shanghai, China). 2’-deoxy-5-methylcytidine (5mdCyd) (99%) was purchased from Aladdin (Shanghai, China). Further purification was taken for 5mC and dCyd via recrystallization and the 7



TA signal from recrystallized samples matches well with that from as received samples (Figure S15 and S16). 18.2 MΩ deionized water (Direct-Q3 UV, Merck Millipore) was use for preparation of phosphate buffer solution (200 mM, pH=7.4). Acetonitrile was also purchased from Sigma-Aldrich (USA) (99.8% anhydrous) and Richjoint chemical Ltd. (Shanghai, China) (≥99%). Steady-state UV/Vis absorption spectra (Figure 1、S2、S3、S11) were measured using a double beam UV-Vis spectrometer (TU1901, Beijing Purkinje General Instrument Co. Ltd.). All spectra were background corrected. Transient absorption (TA) spectra and kinetics were measured using femtosecond transient absorption spectrometer (Helios fire, Ultrafast System). The fundamental pulses were generated with a Ti: Sapphire laser system (Astrella, 800 nm, 100 fs, 7 mJ/pulse, and 1 kHz repetition rate, Coherent Inc.). White light continuum (WLC) probe beam was generated by focusing the fundamental beam into a CaF2 or Sapphire window and the time window limit is 8 ns. A fraction of the fundamental beam was used to produce pump beams via an optical parametric amplifier (OPerA Solo, Coherent Inc.). TA kinetics in the UV region was measured by a previous reported home-built pump-probe setup46. The 266 nm beam was generated from the third harmonic of the fundamental beam; another beam was generated from optical parametric amplifier. They both can be used as pump or the probe beam and the time window limit is 1.5 ns. All experiments were carried out at room temperature. A 2 mm fused silica cuvette was used in TA experiments. This cuvette holds up ~750 µl solution for an experiment. In order to prevent sample degradation, a small magnet 8


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stirred is used during TA experiment. Steady-state absorption of the samples were measured before and after TA experiment, the absorption value changed no more than 3%. The intensities used in our experiments is low enough (

Excited State Decay Pathways of 2â²-Deoxy-5-methylcytidine and

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