Decay Pathways of Thymine Revisited - The Journal of Physical

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Decay Pathways of Thymine Revisited Bert Manuel Pilles, Benjamin Maerz, Jinquan Chen, Dominik B. Bucher, Peter Gilch, Bern Kohler, Benjamin P. Fingerhut, Wolfgang Zinth, and Wolfgang J. Schreier J. Phys. Chem. A, Just Accepted Manuscript • DOI: 10.1021/acs.jpca.8b02050 • Publication Date (Web): 11 May 2018 Downloaded from http://pubs.acs.org on May 13, 2018

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The Journal of Physical Chemistry

Decay Pathways of Thymine Revisited Bert M. Pilles1, Benjamin Maerz1, Jinquan Chen2, Dominik B. Bucher1, Peter Gilch3, Bern Kohler4, Wolfgang Zinth1*, Benjamin P. Fingerhut5, Wolfgang J. Schreier1* 1

Lehrstuhl für BioMolekulare Optik, Fakultät für Physik and Munich Center for Integrated Protein

Science CIPSM, Ludwig-Maximilians-Universität München, Oettingenstr. 67, 80538 München, Germany 2

State Key Laboratory of Precision Spectroscopy, East China Normal University, 3663 North

Zhongshan Road, Shanghai 200062, China 3

Institut für Physikalische Chemie, Heinrich-Heine-Universität Düsseldorf, Universitätsstr. 1, 40225

Düsseldorf, Germany 4

Department of Chemistry and Biochemistry, The Ohio State University, 100 W. 18th Ave., Columbus,

OH 43210, USA 5

Berlin, Germany.

, Max-Born-Str. 2A, D-12489

ABSTRACT: The decay of electronically excited states of thymine (Thy) and thymidine 5’monophosphate (TMP) was studied by time-resolved UV/vis and IR spectroscopy. In addition to the well-established ultrafast internal conversion to the ground state, a so far unidentified UV induced species is observed. In D2O this species decays with a time constant of 300 ps for thymine and of 1 ns for TMP. The species coexists with the lowest triplet state and is formed with a comparably high 1 ACS Paragon Plus Environment

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quantum yield of about 10 % independent of the solvent. The experimentally determined spectral signatures are discussed in the light of quantum chemical calculations of the singlet and triplet excited states of thymine.

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INTRODUCTION DNA exhibits ultrafast radiationless deactivation channels for electronically excited states. Decay occurs via internal conversion on time scales of only a few hundred femtoseconds 1. Most of the electronic excess energy is transformed into vibrational motion and effectively taken away from the DNA to the surroundings with typical cooling times of a few picoseconds in aqueous solution 1, 2. The observation of these channels led to the notion that the building blocks of life were selected because ultrafast energy dissipation suppresses UV-induced damage to the genetic code3. Yet, there is also evidence for longer-lived electronic excitations. In base pairs, single strands and double stranded DNA, the spatial organization of the bases creates new deactivation channels that are not found in monomers and cover multiple time scales, e.g. charge transfer excited states with pico- to nanosecond lifetimes have been detected

3-9

. Notably, longer lived electronic excitations which are “

” have also been

proposed for monomeric DNA bases 1. Here, the pyrimidine base thymine is of special interest. It has been shown that the main UV-induced photolesion between neighboring thymine bases, the cyclobutane thymine dimer or CPD lesion, can be formed in an ultrafast reaction (within 1 ps) if at the instant of UV absorption neighboring thymine bases are close enough for the reaction to occur

10, 11

. Additionally the population of long lived triplet

states of thymine has been demonstrated, which could in principle permit damage formation during the entire lifetime of the triplet state

12

. Triplet states of thymine are believed to be formed via excited

singlet n* states, which have also been mentioned as potential precursors for other DNA photolesions, namely pyrimidine photohydrates and the pyrimidine (6-4) pyrimidone photoproducts 13. Yet, there has been considerable controversy concerning the role of 1n* excited states of thymine. Conical intersections (CIs) between the initially populated 1* state and the ground state have been located (cf. ref

14-16

) and simulations in the gas phase suggest that the 1nπ* state constitutes an

additional ultrafast relaxation channel together with ultrafast population of triplet states 3 ACS Paragon Plus Environment

17

. Based on


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femtosecond transient absorption experiments in the UV range, Hare et al. proposed that bifurcation leads to two decay pathways of the electronically excited state, a subpicosecond internal conversion channel and a second channel that contains passage through a dark intermediate state assigned to an n* excitation with a lifetime of about 130 ps for thymidine 5`-monophoshate (TMP)

13

. A small

fraction of the singlet n* population is suggested to undergo intersystem crossing to the lowest triplet state in competition with internal conversion to the electronic ground state. The same scheme has very recently been used to explain TR R

m

2’-deoxythymidine (dT) dissolved in chloroform 18.

However, other studies yielded different results concerning the lifetime and existence of excited n* states. Kwok et al. performed a combined broadband time-

v

ﬂ

absorption study on thymidine in the UV and visible spectral range and did not observe a 100 ps decay component 19. Instead it was proposed that the singlet n* state of thymidine has a lifetime of only 760 fs and acts as a doorway state towards the triplet state. This process is supposed to occur in competition with the repopulation of the ground state. A similar mechanism has been suggested by Prokhorenko et al. with a decay time of 1.7 ps for the singlet n* state of thymine

15

. Time-resolved fluorescence

experiments on thymine in water yielded a biexponential decay with time constants of 0.2 ps and 0.6 ps 20

. In a recent study of the ultrafast dynamics of thymine, it was proposed that a time constant of less

than 100 fs can be assigned to the relaxation from the first excited S2 1(* state to the S1 1(n* state while a time constant of 1 ps can be assigned to the relaxation from a deformed S 2d 1(* state to the S0 ground state 14. Also, gas-phase experiments support a rapid internal conversion between the initially excited 1* and 1n* states of thymine 21, 22. Meanwhile, a recent study on the excited-state relaxation of hydrated thymine and thymidine using liquid-jet photoelectron spectroscopy suggested that an n* state is not involved in the relaxation process of thymine and thymidine in aqueous solution at all 23.


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Scheme 1. Chemical structures of thymine (Thy) and thymidine 5’-monophosphate (TMP).

These sometimes contradictory results seem to be related to the fact that some of the assignments are based solely on transient signals observed at single wavelengths without including a full spectral analysis of the decay associated spectra. The latter has been shown to be a special advantage of time resolved vibrational spectroscopy in the fingerprint region of the DNA molecules

2, 10

. To resolve the

controversies concerning the observed decay times and state assignments of thymine excited states we recorded transient absorption signals for thymine (Thy) and the thymidine 5’-monophosphate (TMP) after UV excitation (chemical structures of the samples are given in scheme 1) in different solvents. The transient experiments cover the UV, the visible and the infrared spectral range. The experiments are discussed in the light of quantum chemical calculations focusing on the IR spectral signatures of thymine excited states, notably the lowest triplet * state and singlet or triplet n* states.

EXPERIMENTAL AND THEORETICAL METHODS Sample Preparation. Thymidine 5’-monophosphate (TMP) and thymine (Thy) were purchased from Sigma Aldrich. The samples were obtained as lyophylized powders and used without further purification. The pure solvents D2O, methanol-d4 (CD3OD) and acetonitrile-d3 (CD3CN) were obtained from Sigma Aldrich as well. To the D2O solvent phosphate buffer (50 mM, pD ~ 7) was added. 5 ACS Paragon Plus Environment


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Steady-State Experiments. Steady-state absorption spectra in the UV/Vis were recorded using a Perkin-Elmer Lambda 750 spectrophotometer. IR spectra were recorded using a Fourier transform infrared (FTIR) spectrometer (Bruker IFS 66).

Time-Resolved Experiments in the UV. Transient absorption (TA) signals were recorded using a 1 kHz amplified Ti:Sapphire laser system describedearlier.24 In general, pump pulses with a central wavelength of 267 nm were generated via third harmonic generation using a small portion (20%) of the m

800 m. T

1 μJ

m

w

333 H by m

(New Focus) positioned in front of the sample to modulate the pump beam. A second portion (40%) of the fundamental output was used to pump an optical parametric amplifier (OPerA solo, Coherent Inc.). The probe pulses were obtained by second harmonic generation of the sum-frequency mixing signal from the OPerA solo. By using two UV-grade polarizers placed in pump and probe beam path before the sample, the angle between the linear polarizations of pump and probe pulses was precisely set to parallel, perpendicular and magic angle (54.7°). Pump and probe spot diameters were measured by knife-edge scan at the sample position to be 0.48 mm and 0.15 mm, respectively. After the sample, the probe pulses were spectrally isolated by a monochromator and detected by a photomultiplier tube read out by a lock-in amplifier (SR830, Stanford Research Systems) that was referenced to the mechanical chopper. Signals from the lock-in amplifier were recorded by data collection software written in Labview (National Instruments). A detailed analysis of the influence of polarization conditions and corresponding signals in the transient data due to rotational-diffusion is given in the SI, section 3. All solutions for TA experiments were prepared to have a ground state absorbance of 1.0 ± 0.02 in a path length of 1.0 mm at the pump wavelength of 267 nm. TA measurements were performed using a home-built flow cell system at room temperature. In this system, the solution under study was re6 ACS Paragon Plus Environment

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circulated using a peristaltic pump (model Masterflex7524-00, Cole-Parmer) through a flow cell (model TFC-M25-3, Harrick Scientific Products) made from two 1 mm thick CaF2 windows separated by a 1 mm Teflon spacer.

Time-Resolved Broadband Experiments in the UV/Vis and IR. The broadband measurements in the UV/vis and IR spectral range were performed using femtosecond pulses from a Ti-sapphire laseramplifier system as described in ref.

25

. In brief, the instrument is based on a Ti:saphire CPA system

(Spitfire Pro XP) yielding 100 fs pulses at 800 nm with a repetition rate of 1kHz. For excitation at 266 nm the third harmonic of the fundamental was used. Excitation pulses at 250 nm were obtained by frequency doubling the output of a non-collinear parametric amplifier resulting in an excitation energy of 0.8 µJ at a diameter of about 150 µm (fwhm). To suppress the occurrence of two-photon ionization of solvent and solute molecules the excitation pulses were stretched to a duration of about 1.5 ps by passing through 20 cm of fused silica. To reduce common mode noise every second pump pulse was blocked by a mechanical chopper when recording the pump induced absorption changes of the sample. All measurements were performed under magic angle conditions. Probing in the UV/Vis spectral range was performed by using a suitably delayed white light continuum (350 nm bis 700 nm) generated in a CaF2 crystal (beam diameter 50 µm at the sample) 26. The spectrum of the transmitted light was recorded by a multi-channel detection system consisting of a concave grating from Zeiss (blazed at 225 nm) for light dispersion and a NMOS linear image sensor (Hamamatsu 14-S3902-512Q in combination with a Tec5 DZA-S3902-4 1M preamplifier electronic). The excitation pulses at 266 nm had an energy of 1 µJ at a diameter of about 160 µm (fwhm). Transient signals from the neat solvent were recorded and used for background correction (see SI). The samples were held in flow cells (pathlength 200 µm, Quartz windows), with a sufficient flow rate to exchange the sample volume between consecutive excitation pulses. 7 ACS Paragon Plus Environment


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IR probe pulses were generated using a combination of a collinear and a non-collinear optical parametric amplifier with subsequent difference frequency mixing in a AgGaS2 crystal. The resulting mid-IR pulses (probe and reference) were spectrally dispersed (Bruker Chromex 250is) and detected by two 64-Channel MCT arrays (Infrared Associates Inc.) connected to a multichannel data acquisition system (IR 0144, Infrared Systems Development Corp.). Excitation was done using UV pulses with an energy of 2 µJ at 266 nm and 0.8 µJ at 250 nm (diameter about 160 µm (fwhm)). The samples were held in flow cells with a pathlength of ~100 µm using BaF2-windows. The sample concentrations used in the IR experiments were 10 mM for TMP in D2O and CD3OD and 10 mM and 3 mM for Thy in D2O and CD3CN, respectively.

Calculations. For the electronic structure calculations the wave-function based complete active space self consistent field method is augmented by multi-reference perturbation theory (14 electrons in 10 active orbitals, equal state averaging over 5 states, level shift = 0.3, multi-state MRPT2: sa5-MSMRPT2/CAS(14/10)/6-31G*; mp2 equilibrium geometry with Cs symmetry) and serves as reference and benchmark for TD-DFT calculations (coulomb attenuated exchange correlation functional: CAMB3LYP

27

). Gas phase optimization of the

1

π*

3

π*

v

b

m

MRPT2/CAS(10/8)/6-31G* level of theory without symmetry restrictions where we have verified that the reduced active space yields the correct state ordering at the Franck-Condon (FK) geometry. Frequencies of C=O marker bands in the excited state are calculated by a mode tracking procedure with the Akira28 program coupled to Molpro 29 (see http://www.molpro.net) with a home-written interface 30 (sa2-MS-MRPT2/CAS(10/8)/6-31G*). Excited state absorption (ESA) from the relaxed 1 π* minimum geometry has been calculated on the sa11-MS-MRPT2/CAS(14/10)/6-31G* level of theory considering all possible interstate excitations. Transition moments between exited states are evaluated on the 8 ACS Paragon Plus Environment

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CAS(14/10)/6-31G* level of theory and rotated by the respective MS-MRPT2 mixing coefficients. Solvent effects on thymine excited states were investigated in a cluster based approach where the starting configuration was taken from a snapshot of a classical MD simulation of thymine in a cubic water box of 18.3 Å (amber force field, SPC/E water model, 20 ps equilibration) followed by classical minimization. Optimization prior to TDDFT simulations was performed to assure a minimum configuration of solute and solvent molecules for excited state calculations. The considered thyminewater cluster comprise all water molecules within 2, 3.5, 5, 6, 7 and 8 Å of the central thymine (CL-2, CL-3.5, CL-5, CL-6, CL-7, CL-8) and consist of 4, 22, 52, 78, 102 and 134 water molecules, respectively. The excited states of the CL-X are evaluated on TD-CAMB3LYP/6-31G* level of theory for CL-2, CL-3.5 and CL-5, while larger clusters are treated on FMO-TD-CAMB3LYP/6-31G* level of theory (FMO1-TD

28

, FMO2-TD

31

). All calculations consider 4 excited roots. All TD-DFT

calculations have been performed within the random-phase approximation as the Tamm-Dankoff (TDA) approximation turned out to produce unreliable results in the full cluster calculation. Optimization of CL-3.5 in various electronic states has been performed on the full TD-CAMB3LYP level of theory. MRPT2/CAS calculations have been performed with Molpro 29, (FMO)-TD-DFT with GAMESS 32, 33, classical MD calculations with NWchem34.

EXPERIMENTAL RESULTS The results of time-resolved transient absorption measurements in the UV and visible spectral range for TMP in aqueous solution are shown in Figure 1. With excitation at 267 nm and probing at 250 nm (Figure 1a), a negative induced absorbance signal is observed that falls within the strong, lowest-energy 1

* absorption band of TMP. The data show a recovery of the initial bleach signature within several

picoseconds and a further signal decrease on the hundred ps to ns time scale. Using a sum of two exponentials and an offset, the data are well reproduced by time constants of 4 ps and 1 ns. The actual 9 ACS Paragon Plus Environment


depopulation of the excited 1* state is expected to occur on the sub-picosecond time scale

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35

.

Therefore, the initial recovery of the signal can be assigned to the repopulation of the electronic ground state (black) including cooling dynamics of hot ground state molecules after ultrafast internal conversion. A residual offset is expected due to the formation of a small amount of long-lived triplet states 36. Data from a broadband transient absorption experiment with excitation at 266 nm and white-light probing are shown in Figure 1b for selected wavelengths (for the full data set, see SI Figure S1). In this experiment the excitation pulses were stretched to about 1.5 ps in order to minimize effects due to twophoton ionisation, therefore sub-ps decay kinetics cannot be resolved. At 375 nm (red), the initially observed signal is dominated by stimulated emission, which results in negative absorbance signals for probe wavelengths below 450 nm. The negative absorption signal is followed by a strong signal increase within 20 ps. Later on the signal decays with a time constant of 1 ns in correspondence with 250 nm probing. At 475 nm (blue) an essentially constant offset is observed. At 550 nm (green) an induced absorption signal is seen that decays within 5 ps to the base line. This signal can be assigned to the induced absorption of the 1* excited state 13.

Figure 1. Transient absorption signals recorded for the nucleotide TMP in D2O. a) Transient absorption signals recorded for excitation at 267 nm and probing at 250 nm. b) Transient absorption signals from a 10 ACS Paragon Plus Environment

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broadband measurement with excitation at 266 nm and white light probing. Fits with time constants of a few picoseconds, describing the initial dynamics including vibrational cooling, and a time constant of 1 ns are given as solid lines. Note that the data in panel b) have been corrected for the signal of solvated electrons created by two-photon ionization of the solvent (see SI Figure S2).

A quantitative evaluation of the broadband dataset including a global fit is given in the SI (cf. Figure S1). The fit has been performed from 10 ps onwards to exclude the contributions from vibrational cooling. The decay associated difference spectra (DADS) reveal that the species that decays with 1 ns exhibits an induced absorption peaking around 350 nm. The offset spectrum is characterized by a broad absorption between 350 nm and 550 nm. The latter is found to be in reasonable agreement with the absorption found for the lowest triplet state (3*) of TMP 37. The signals of the 1 ns decay component and the offset are very broad and overlap, which hampers a clear spectral assignment based on the UV and visible data alone. To overcome these limitations we performed time resolved measurements with probing in the IR spectral range.



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Figure 2. (a, b) Broadband transient absorption signals in the IR recorded for TMP in D2O (left) and methanol-d4 (right) after UV-excitation at 266 nm. The data in D2O have been corrected for the signature of solvent heating (see SI section 2.1). (c, d) Transient spectra at the indicated delay times. The ground state absorption spectrum of TMP is given in gray for comparison. (e, f) Decay associated difference spectra (DADS) obtained from a global-fit yielding time constants of  = 1 ns and  = 1.5 ns (red) for species X in D2O and methanol-d4, respectively. The residual difference spectrum remains as an offset and can be assigned to the lowest triplet state (black).

Figure 2 shows the results from time resolved infrared experiments on TMP in D2O and methanol-d4 (CD3OD) after excitation at 266 nm. The first few picoseconds are dominated by cooling signatures of 12 ACS Paragon Plus Environment

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hot ground state molecules after ultrafast internal conversion (see panels a and b in Figure 2). The initially hot ground state molecules show strong bleach signals at the position of the ground state absorption bands and induced absorption to lower frequencies. These signatures arise from anharmonic coupling of the observed ground state vibrational modes to excited low-frequency modes and decay with the cooling of the molecules within the first 10 picoseconds

10, 38, 39

. The ground state absorption

spectrum of TMP is given in panel c (gray). The prominent absorption bands can be assigned as follows (cf. ref. 40) : (i) 1630 cm-1, ring vibrational mode including the C5=C6 double bond; (ii) 1661 cm-1, mainly C4=O8 double bond stretching; (iii) 1681 cm-1, mainly C2=O7 double bond stretching. After the cooling signatures have diminished, two characteristic absorption features are observed in the transient difference spectra (see Figure 2 c,d): (i) a prominent induced absorption located between 1730 cm-1 and 1760 cm-1; (ii) an induced absorption band peaking somewhat below 1600 cm-1. While the former signal is clearly observed in both solvents, the later is rather small for TMP in D2O but is observed as a prominent feature in CD3OD (see Figure 2 a,b). Performing a quantitative analysis of the time resolved data results in time constants of 1 ns (TMP in D2O) and 1.5 ns (TMP in CD3OD) for the decay of the prominent feature peaking at 1740 cm-1 and a long-term offset, which includes the absorption feature at 1600 cm-1. The corresponding decay associated difference spectra (DADS) are given in panels e and f of Figure 2. The signature of the long term offset matches the absorption difference spectrum found for the lowest triplet state of thymine identified in a recent time resolved IR study on TMP and (dT)18 6. Thus, the IR data agree with the results obtained in the UV/Vis spectral range. In both experiments, two different species are observed: the lowest triplet state and a second species, which we will refer to as species X. While the triplet state is responsible for the long term offset, species X decays on a 1 ns time scale. Additionally the data of TMP in CD3OD clearly show that the decay of species X on the 1 ns time scale is not accompanied by a buildup of the lowest triplet state (see triplet marker band around 1600 cm-1 in panels b and d of 13 ACS Paragon Plus Environment


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Figure 2). Instead, the data indicate strongly that the full amount of species X with its characteristic absorption at 1750 cm-1 is present within 1 ps after UV excitation whereas the triplet state absorption at 1600 cm-1 shows some delayed increase and is fully formed only within about 10 ps. A comparison of transients at 1600 cm-1 and 1750 cm-1 indicative of the corresponding species is given in SI Figure S4 for TMP in CD3OD. A similar delayed onset of triplet state absorption has been observed by Röttger et al. for dT in chloroform

18

and in acetonitrile

41

. Thereby, it has to be noted that the monitored

absorption increase occurs on the same time scale as vibrational cooling and cannot be distinguished from anharmonic frequency shifts of an initially hot 3* state in analogy to the anharmonic shifts observed for the hot ground state molecules after ultrafast IC.

Table 1. Lifetimes (Xand Quantum Yields (X) of Species X Retrieved from Transient Absorption Measurements on Thymine (Thy) and Thymidine 5’-Monophosphate (TMP) a sample

solvent

X /ns

X

3*

Thy

D2O

0.3

0.075

p* Blue-shift Phenomenon1. J. Am. Chem. Soc. 1955, 77, 4462-4468. 57. Etinski, M.; Marian, C. M., Ab Initio Investigation of the Methylation and Hydration Effects on the Electronic Spectra of Uracil and Thymine. Phys. Chem. Chem. Phys. 2010, 12, 4915-4923. 28 ACS Paragon Plus Environment

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58. Elsayed, M. A., Spin - Orbit Coupling and the Radiationless Processes in Nitrogen Heterocyclics. J. Chem. Phys. 1963, 38, 2834. 59. Turro, N. J.; Ramamurthy, V.; Scaiano, J. C., Modern Molecular Photochemistry of Organic Molecules. University Sciences Books, Sausalito, California: 2010. 60. Heinz, B.; Schmidt, B.; Root, C.; Satzger, H.; Milota, F.; Fierz, B.; Kiefhaber, T.; Zinth, W.; Gilch, P., On the Unusual Fluorescence Properties of Xanthone in Water. Phys. Chem. Chem. Phys. 2006, 8, 3432-3439. 61. Rai-Constapel, V.; Villnow, T.; Ryseck, G.; Gilch, P.; Marian, C. M., Chimeric Behavior of Excited Thioxanthone in Protic Solvents: II. Theory. J. Phys. Chem. A 2014, 118, 11708-11717. 62. Torres Ziegenbein, C.; Fröbel, S.; Glöß, M.; Nobuyasu, R. S.; Data, P.; Monkman, A.; Gilch, P., Triplet Harvesting with a Simple Aromatic Carbonyl. Chem. Phys. Chem. 2017, 18, 2314-2317. 63. Villnow, T.; Ryseck, G.; Rai-Constapel, V.; Marian, C. M.; Gilch, P., Chimeric Behavior of Excited Thioxanthone in Protic Solvents: I. Experiments. J. Phys. Chem. A 2014, 118, 11696-11707. 64. Onidas, D.; Markovitsi, D.; Marguet, S.; Sharonov, A.; Gustavsson, T., Fluorescence Properties of DNA Nucleosides and Nucleotides: A Refined Steady-State and Femtosecond Investigation. J. Phys. Chem. B 2002, 106, 11367-11374. Table of Contents


O

H3C 1 2 3 4

O 8 Journal of Physical Chemistry The Page 30 of 35 H C 3

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4

3 NH

6 1 2

NH

O HO

P

N

O

O O7 N OH ACS Paragon Plus Environment H

Thy

OH

O

TMP

∆A / mOD

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DADS(τ1=1 ns)

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Decay Pathways of Thymine Revisited - The Journal of Physical

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