Direct Observation of Triplet-State Population Dynamics in the RNA

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Letter

Direct Observation of Triplet-State Population Dynamics in the RNA Uracil Derivative 1-Cyclohexyluracil Matthew M Brister, and Carlos E. Crespo-Hernández J. Phys. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acs.jpclett.5b01901 • Publication Date (Web): 21 Oct 2015 Downloaded from http://pubs.acs.org on October 22, 2015

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The Journal of Physical Chemistry Letters

Direct Observation of Triplet-State Population Dynamics in the RNA Uracil Derivative 1-Cyclohexyluracil Matthew M. Brister and Carlos E. Crespo-Hernández* Department of Chemistry and Center for Chemical Dynamics, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106 Corresponding Author * [email protected]

ACS Paragon Plus Environment

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ABSTRACT

Investigation of the excited-state dynamics in nucleic acid monomers is an area of active research due to the crucial role these early events play in DNA and RNA photodamage. The dynamics and rate at which the triplet state is populated are key mechanistic pathways yet to be fully elucidated. Direct spectroscopic evidence is presented in this contribution for intersystem crossing dynamics in a uracil derivative, 1-cyclohexyluracil. It is shown that intersystem crossing to the triplet manifold occurs in one picosecond or less in acetonitrile solution—at least an order of magnitude faster than previously estimated experimentally. Broadband transient absorption measurements also reveal the primary electronic relaxation pathways of the uracil chromophore, including the absorption spectra of the 1*, 1n*, and 3* states and the rates of vibrational cooling in the ground and 3* states. The experimental results are supported by density functional calculations.

TABLE OF CONTENTS IMAGE S0,hot

1n*

A SE

3*

1*

KEYWORDS DNA and RNA Monomers, Transient Absorption Spectroscopy, Excited States, Excited-State Dynamics, Photochemistry, DFT Calculations


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Interest in the photochemistry of DNA and RNA has been a longstanding motivation for investigating the excited-state dynamics in nucleic acid monomers from both experimental and computational perspectives.1-15 A key mechanistic aspect of this chemistry that is poorly understood and often overlooked12 is the participation of the triplet state and the rate at which singlet-to-triplet population transfer occurs. This is surprising given that the long-lived triplet state is thought to play an important role in the photochemistry of the pyrimidine monomers in dilute solutions,16-19 as well as in DNA oligomers,14,

20-22

and has long been recognized to be

involved in the formation of the pyrimidine cyclobutane dimers.16-19, 23-28 Hare et al.30 presented the first experimental evidence of the population of the triplet state in a RNA pyrimidine monomer on a sub-10 ps time scale in solution. Fast population of the triplet state is expected because intersystem crossing must occur within a few picoseconds to compete with the observed ultrafast internal conversion to the ground state in the DNA and RNA pyrimidine monomers. In fact, experimental12,

21, 31-33

and computational11,

34-37

evidence

supporting the idea that intersystem crossing occurs on an ultrafast time scale is accumulating. However, contrary to the significant progress achieved in understanding the electronic relaxation mechanisms that make DNA and RNA photostable,1-8,

10-12, 14

the mechanism by which

intersystem crossing to the triplet manifold occurs is poorly understood and direct spectroscopic measurements of the intersystem crossing rates are needed. This gap in spectroscopic information is primarily due to the fact that the absorption spectra of the excited-state species involved overlap strongly and the yields of triplet-state population are relatively small for the DNA and RNA pyrimidine monomers in solutions.12 In this letter, we present direct spectroscopic evidence of sub-picosecond intersystem crossing to the triplet manifold in a uracil derivative by using broadband transient absorption


3


spectroscopy and an in-depth global and target analysis of the observed kinetics. In particular, we investigated the excited-state dynamics of 1-cyclohexyluracil (1CHU, Figure 1) in acetonitrile and show that intersystem crossing to the triplet manifold occurs in less than one picosecond. 7 6 5

(103 M-1cm-1)

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4 3 2 1 0 200

225

250

275

300

325

350

Wavelength (nm)

Figure 1. Molar absortivity spectrum of 1-cyclohexyluracil in acetonitrile. Inset: Optimized structure in the ground state at the B3LYP/IEF-PCM/6-311++G(d,p) level of theory.

Figure 2 shows the transient absorption spectra of 1CHU after excitation at 270 nm, whereas Figure 3 shows representative kinetic traces and the decay associated spectra of the primary transient absorption species involved (see the SI for details). The transient data reveal a complex spectral and temporal overlap of absorption bands, which is associated with the presence of multiple transient species. Specifically, at least five different absorption bands are observed in the spectral window from 325 to 700 nm (Figs. 2 and 3b). Two of these absorption bands have maxima at wavelengths shorter than 340 nm (one with negative amplitude and the other with positive amplitude), whereas the other three bands have maxima at 365  10, 410  10 and 525  2 nm. Initially, the negative-amplitude absorption band at probe wavelengths shorter


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than 340 nm and the positive-amplitude absorption band with maximum at 525 nm decay simultaneously, displaying an apparent isosbestic point at 465 ± 3 nm (Fig. 2a). The simultaneous decay of these absorption bands leads to the formation of a broad transient absorption spectrum that covers the full spectral probe window (see spectrum at ~0.60 ps in Fig. 2a). This broad spectrum can be divided arbitrarily in two spectral regions by using the apparent isosbestic point at 445  2 nm as a reference probe wavelength. The transient signal at wavelengths shorter than 445 nm decays within 3 ps (Fig. 2b), whereas the transient signal at longer wavelengths persists until about 20 ps. Hence, these two spectral regions are associated with two different relaxation processes. As the population in these two channels decay, two additional absorption bands are observed with adjacent maxima at 365  10 and 410  10 nm (Figs. 2b and 3b). The absorption at 365 and 410 nm of these two overlapping bands begins to decay within the 3 ns temporal window of our experimental setup, whereas that at 575 nm does not decay appreciably within this time window (see Fig. S1 and additional discussion in SI).


5


-3 A (10 )

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Time delay (ps): 0.00 0.17 0.27

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-3 A (10 )

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400

(b)

450 500 550 600 Wavelength (nm)

650

Time delay (ps): 0.60 2.96 20.8

1.5

0.84 6.00

1.27 12.0

0.8

0.0 350

400

450

500

550

600

650

Wavelength (nm)

Figure 2. Transient absorption spectra for 1-cylohexyluracil in acetonitrile at time delays from 0 to 600 fs (a) and from 0.60 to 20 ps (b). The excitation wavelength was set at 270 nm. Time zero was set at the maximum amplitude of the stimulated emission band of 1-cyclohexyluracil around 325 nm. Arrows are included to highlight the primary changes in the absorption bands.

Figure 3a shows representative kinetic traces taken at 330, 365, 410, 525 and 575 nm over the initial 20 ps, whereas Figure S1 shows the decay of the transient signals out to 3 ns. These probe wavelengths were chosen to highlight the relaxation dynamics of the multiple transient absorption species described above (Figs. 2 and 3b). The solid fit lines were obtained from a target and global analysis of the full broadband transient absorption data (see SI), convoluted with an instrument response function of 200  50 fs. The following lifetimes were obtained from this analysis: 1 = 240  50 fs; 2 = 400  60 fs; and 3 = 5  1 ps. The first


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lifetime is associated with the simultaneous decay of the negative-amplitude absorption band and the band with an absorption maximum around 525 nm (Figs. 2a and 3). The second lifetime is associated with the relaxation of the transient signal at wavelengths shorter than 445 nm and the third lifetime is associated with the decay of the transient signal above this probe wavelength (Figs. 2b and 3b). (a)

(10-3)

2

1

0 330 nm 525 nm

-1

5

(10-3)

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0

1

(b)

2

365 nm 575 nm

410 nm

4 6 8 10 12 14 16 18 20 Time (ps) 1* (Vis) + SE (UV)

4

 S0,hot (UV) + 3*hot (Vis)

3

 3*hot (Vis) + S0,hot (UV)  1n*

2

residual (x 5): 3*

1 0

-1 -2 -3

350

400

450 500 550 Wavelength (nm)

600

650

Figure 3. (a) Kinetic decay traces of 1cyclohexyluracil in acetonitrile at selected probe wavelengths. The excitation wavelength was set at 270 nm. (b) Decay associated spectra obtained using a target analysis method, in which a sum of four exponential components plus a constant offset were used to globally-fit the broadband data across the 3 ns time window. As highlighted in the legend, the DAS show some residual contribution from the absorption spectra of other


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transient species, which is due primarily to the strong overlap of the transient absorption species in the spectral probe windows used (see SI for details).

We now use the extensive results from previous transient absorption experiments for 1CHU in protic and aprotic solvents at discrete probe wavelengths30 and the vertical excitation energies reported below (Fig. 4) to assign the broadband transient absorption spectra reported in Figures 2 and 3b. The negative-amplitude absorption band is assigned to stimulated emission from 1CHU based on two observations: (1) the ground-state absorption spectrum does not absorb above 300 nm in acetonitrile (Fig. 1), ruling out ground-state depopulation; and (2) a negligibly small fluorescence emission band is observed in the same spectral region for 1CHU in acetonitrile (Fig. S2). Gustavsson et al.40 reported that the fluorescence emission spectrum of uracil has maximum around 320 nm in acetonitrile, which further supports the assignment of this band to stimulated emission in the transient spectra of 1CHU. The positive absorption band with maximum at 525 nm decays in lockstep with the stimulated emission band and is therefore assigned to excited-state absorption of the 1ππ* state. As the 1ππ* state decays with a lifetime of 240 ± 50 fs, a broad absorption spectrum develops that covers the full spectral window from ~320 to 700 nm at ~0.60 ps (Fig. 2a). As mentioned above, the transient signal at wavelengths shorter than 445 nm decays on a faster time scale than the transient signal at longer probe wavelengths. Hence, they should be associated to two distinct relaxation processes. According to the target and global fit analysis, the absorption band at wavelengths shorter than 445 nm decays with a lifetime of 400 ± 60 fs, whereas that at longer wavelengths decays with a lifetime of 5 ± 1 ps. We assign the high-energy band below 445 nm to vibrational cooling dynamics in the ground state, whereas the lower-energy transient


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signal is assigned to vibrational cooling dynamics in the 3ππ* state. The weaker solvent hydrogen bonding interactions with the 3ππ* state than with the ground state likely explains the large difference in these vibrational cooling lifetimes, as proposed previously.30 In this regard, the two values represent average vibrational cooling lifetimes obtained from the global fit analysis of the full broadband data. The population of the vibrationally-hot 3ππ* state is further supported by the vertical excitation energies reported in Figure 4, which show that 1.6 eV of excess energy is available when the population in the 1ππ* state (or 1nπ* state) intersystem crosses to populate the triplet manifold in acetonitrile. In addition, the absorption spectrum of the 3ππ* state has a maximum around 410 nm with an absorption tail extending out to 650 nm, as describe below, further supporting its assignment to vibrational cooling dynamics in the 3ππ* state. As vibrational cooling dynamics cease within the initial ~20 ps, two additional long-lived absorption bands are observed with maxima at 365 and 410 nm, respectively, that strongly overlap with one another (Fig. 3b). A significant fraction of the absorption band with maximum at 365 nm decays within a few nanoseconds. This leaves a residual absorption band with maximum at 410 nm that does not decay significantly within the 3 ns time window. Due to the close proximity of the two absorption maxima, the difference in the dynamics of these two transient species are most readily apparent from a comparison of the kinetic traces taken at 365 and 575 nm (Fig. S1), as the 410 nm band has an absorption tail that extends out to 650 nm (Fig. 3b). The transient species with maximum at 410 nm is assigned to the relaxed 3ππ* state, whereas the shorter-lived absorption band at 365 nm is assigned to population that did not intersystem cross and rather reached the relaxed 1nπ* state (see SI for further discussion). This assignment is in agreement with the results from previous transient absorption studies.30 The


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absorption band at 410 nm can be quenched by molecular oxygen,30 further supporting its assignment to the 3ππ* state. Further support for the relaxation pathways proposed above comes from a closer look at the calculated vertical excitation energies for the relevant singlet and triplet exited states reported in Figure 4. Excited-state calculations have not been reported in the literature for 1CHU. The energy values and oscillator strengths are compared to those calculated for uracil and reported in Table S1. It is apparent that there is a 1nπ* state lower in energy than the 1ππ* state in vacuum, whereas the two states are isoenergetic in acetonitrile. The calculations also predict that the lowest-energy triplet state is a 3ππ* state in both vacuum and acetonitrile. There are two additional triplet states with ππ* and nπ* character below the vertically-excited 1ππ* state. The energy gaps of these two 3ππ* and 3nπ* states decreases in going from vacuum (0.3 eV) to acetonitrile (0.1 eV), but their energetic ordering remains the same.

1

5.0 1

*

1

n*

3

* & 1n*

4.5

3

* n*

3

*

3

Energy (eV)

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n*

4.0

3.5 3

3

*

*

3.0 Vacuum

Acetonitrile

Figure 4. Singlet and triplet vertical excitation energies for 1-cyclohexyluracil in vacuum and in acetonitrile calculated at the TD-PBE0/6311++G(d,p)//B3LYP/6-311++G(d,p) level of theory. The IEF-PCM solvation model was used for the vertical excitation energies in acetonitrile (see Methods section in the SI for details).


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According to the vertical excitation energies shown in Figure 4, excitation of 1CHU initially populates the 1ππ* state since the oscillator strength of the 1nπ* state is nearly zero and its magnitude does not change in going from vacuum to acetonitrile (Table S1). Once the 1ππ* state is populated, it can internally convert to the 1nπ* state and the ground state or it can intersystem cross to the triplet manifold directly (i.e., to the 3n* state, according to the propensity rules38-39). Any population reaching the 1nπ* state can internally convert to the ground state or can intersystem cross to the triplet manifold (i.e., both the T3 and T1 states have ππ* character and can potentially be populated37,38). The small energy gap between the singlet and triplet states in the Frank-Condon region, particularly between the S1(n*) and the T3(*) states in vacuum (0.1 eV) and in acetonitrile (0.3 eV), suggests that population of the triplet manifold should be competitive with internal conversion to the ground state. Hence, the calculations shown in Figure 4 are in agreement with the relaxation pathways and the assignment of the transient absorption species discussed above and in Fig. 3b. Evidently, the transient absorption spectra overlap strongly during the initial 20 ps, which limit the prospect of determining an exact intersystem crossing lifetime. However, the transient data presented in Figure 2 clearly show that population of the vibrationally-excited 3ππ* state is observed at a time delay of ~0.60 ps and that the relaxed 3ππ* state begins to resolve in 3 ps or less. Taking these two observations together, the transient data show direct spectroscopic evidence of intersystem crossing to the triplet state in less than one picosecond. In fact, assuming that branching of population to the 1nπ* state and the ground state are the only relaxation pathways depopulating the 1ππ* state with a ca. 240 ± 50 fs lifetime, an intersystem crossing lifetime in the range of 290 to 440 fs can be estimated by using the reported internal conversion


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(66%) or intersystem crossing (34%) yields for 1CHU in acetonitrile (see SI for details).30 This range of intersystem crossing lifetimes support the sub-1 ps intersystem crossing lifetime estimated in this work. The experimental and computational results provide direct evidence that three relaxation pathways occur on an ultrafast time scale: (1) internal conversion to the 1nπ* state; (2) internal conversion to the ground state; and (3) intersystem crossing to the triplet manifold. However, identifying the precise mechanism by which these electronic relaxation processes occur is a more challenging task; a task that requires guidance from high-level static and dynamics simulations. The predictions from the TDDFT calculations reported above, and for uracil in the SI, are consistent with high-level static34, 41-45 and dynamics37, 46-53 calculations performed for uracil in vacuum and in acetonitrile.54-55 Hence, we argue that the results from those calculations can be used with our experimental results to propose an electronic-energy relaxation mechanism for 1CHU. Equally, results for 1CHU could be used to scrutinize the general predictions reached from those high-level calculations for uracil. The quantum-chemical calculations for uracil concur that the 1n* state is the lowestenergy singlet state in the Franck-Condon region, lying below a 1* state, both in vacuum and acetonitrile.10-11, 13, 54 Depopulation of the excited 1* state leads to population of the ground and 1n* states; both relaxation pathways are mediated by corresponding conical intersections. These predictions are in agreement with the ultrafast depopulation of the 1* state in both uracil and 1CHU in ca. 200 fs. The calculations also predict that the excited-state population can intersystem cross to the lowest-energy 3* state: (1) directly from the 1* state; (2) via a 1n* intermediate state; or (3) via a 3n* intermediate state,34, 42-43 in agreement with the calculations presented in Figure 4 and the SI. However, the most recent static and dynamics calculations


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overwhelmingly favor the 1n* state as the doorway state in the intersystem crossing pathway from the 1* state to the 3* state (i.e., a 1*  1n*  3* relaxation pathway).34, 37, 44-45 In fact, intersystem crossing lifetimes as short as sub-2.4 to 5 ps have been estimated from both dynamics37 and static34 calculations, respectively, in semi-quantitative agreement with our experimental results. The predicted lifetimes depend on the relative energy gap between the excited-states involved and on the level of theory used,34, 37 with significant variations in the case of the static calculations (from 5 to up to 100 ps).34 In summary, identifying the mechanism by which the triplet state is populated and the rate at which this process occurs in pyrimidine monomers in solution is crucial because of the leading role that long-lived triplet states play in DNA and RNA photochemistry.14,

17-18

Broadband transient absorption spectroscopy reveals the absorption spectra of the 1*, 1n*, and 3* states; as well as the stimulated emission band and vibrational cooling dynamics in the S0 and 3* states of 1CHU. The ultrafast internal conversion of the 1* state and the subsequent vibrational cooling dynamics in the ground and lowest-energy triplet states have been previously documented by Hare et al.30 What is novel about this work is the observation of intersystem crossing to the 3* state in less than one picosecond. This work presents the first spectroscopic evidence that intersystem crossing to the triplet manifold in the uracil chromophore occurs on the femtosecond time scale. This is more than tenfold faster than previously estimated,30 and is in semi-quantitative agreement with the most recent ab initio molecular dynamics simulations performed for uracil.37 Work is currently underway to determine the intersystem crossing rates in other DNA and RNA pyrimidine derivatives of biological interest. ASSOCIATED CONTENT


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Supporting Information Experimental and computational methods; representative transient absorption decay traces up to 3 ns; steady-state fluorescence emission spectrum of 1-cyclohexyluracil; and singlet- and tripletstate vertical excitation energies and oscillator strengths for 1-cyclohexyluracil and uracil; estimation of the intersystem crossing rate of 1-cyclohexyluracil from a parallel kinetic model. This material is available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. ACKNOWLEDGMENTS The authors acknowledge the CAREER program of the National Science Foundation (Grant No. CHE-1255084) for financial support. REFERENCES 1. Crespo-Hernández, C. E.; Cohen, B.; Hare, P. M.; Kohler, B., Ultrafast Excited-State Dynamics in Nucleic Acids. Chem. Rev. 2004, 104, 1977-2019. 2. de Vries, M. S.; Hobza, P., Gas-Phase Spectroscopy of Biomolecular Building Blocks. Annu. Rev. Phys. Chem. 2007, 58, 585-612. 3. Middleton, C. T.; de La Harpe, K.; Su, C.; Law, Y. K.; Crespo-Hernández, C. E.; Kohler, B., DNA Excited-State Dynamics: From Single Bases to the Double Helix. Annu. Rev. Phys. Chem. 2009, 60, 217-239.


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4. Serrano-Andrés, L.; Merchán, M., Are the Five Natural DNA/RNA Base Monomers a Good Choice from Natural Selection? A Photochemical Perspective. J. Photochem. Photobiol. C: Photochem. Rev. 2009, 10, 21-32. 5. Towrie, M.; Doorley, G. W.; George, M. W.; Parker, A. W.; Quinn, S. J.; Kelly, J. M., PsTRIR Covers All the Bases – Recent Advances in the Use of Transient IR for the Detection of Short-Lived Species in Nucleic Acids. Analyst 2009, 134, 1265-1273. 6. Gustavsson, T.; Improta, R.; Markovitsi, D., DNA/RNA: Building Blocks of Life under UV Irradiation. J. Phys. Chem. Lett. 2010, 1, 2025-2030. 7. Kohler, B., Nonradiative Decay Mechanisms in DNA Model Systems. J. Phys. Chem. Lett. 2010, 1, 2047-2053. 8. Kleinermanns, K.; Nachtigallová, D.; de Vries, M. S., Excited State Dynamics of DNA Bases. Int. Rev. Phys. Chem. 2013, 32, 308-342. 9. Zhao, H.; Liu, K.; Song, D.; Su, H., Physical Quenching in Competition with the Formation of Cyclobutane Pyrimidine Dimers in DNA Photolesion. J. Phys. Chem. A 2014, 118, 91059112. 10. Giussani, A.; Serra-Martí, J.; Roca-Sanjuán, D.; Merchán, M., Excitation of Nucleobases from a Computational Perspective I: Reaction Path. Top. Curr. Chem. 2015, 355, 57-97. 11. Mai, S.; Richter, M.; Marquetand, P.; González, L., Excitation of Nucleobases from a Computational Perspective II: Dynamics. Top. Curr. Chem. 2015, 355, 99-153.


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12. Pollum, M.; Martínez-Fernández, L.; Crespo-Hernández, C. E., Photochemistry of Nucleic Acid Bases and Their Thio- and Aza-Analogues in Solution. Top. Curr. Chem. 2015, 355, 245-327. 13. Improta, R.; Barone, V., Excited States Behavior of Nucleobases in Solution: Insights from Computational Studies. Top. Curr. Chem. 2015, 355, 329-357. 14. Schreier, W. J.; Gilch, P.; Zinth, W., Early Events of DNA Photodamage. Annu. Rev. Phys. Chem. 2015, 66, 497-519. 15.

Crespo-Hernández, C. E.; Martínez-Fernández, L.; Rauer, C.; Reichardt, C.; Mai, S.; Pollum, M.; Marquetand, P.; González, L.; Corral, I., Electronic and Structural Elements That Regulate the Excited-State Dynamics in Purine Nucleobase Derivatives. J. Am. Chem. Soc. 2015, 137, 4368-4381.

16. Fisher, G. J.; Johns, H. E., Pyrimidine Photohydrates. In Photochemistry and Photobiology of Nucleic Acids, Wang, S. Y., Ed. Academic Press: New York, 1976; Vol. 1, pp 169-224. 17. Cadet, J.; Vigny, P., The Photochemistry of Nucleic Acids. In Bioorganic Photochemistry, Morrison, H., Ed. New York, 1990; Vol. 1, pp 1-272. 18. Ruzsicska, B. P.; Lemaire, D. G. E., DNA Photochemistry. In CRC Handbook of Organic Photochemistry and Photobiology, Horspool, W. M.; Song, P.-S., Eds. CRC Press: Boca Raton, 1995; pp 1289-1317. 19.

Cuquerella, M. C.; Lhiaubet-Vallet, V.; Bosca, F.; Miranda, M. A., Photosensitised Pyrimidine Dimerisation in DNA. Chem. Sci. 2011, 2, 1219-1232.


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Figure 1. Molar absortivity spectrum of 1 cyclohexyluracil in acetonitrile. Inset: Optimized structure in the ground state at the B3LYP/IEF-PCM/6-311++G(d,p) level of theory. 62x47mm (600 x 600 DPI)



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Figure 2. Transient absorption spectra for 1-cylohexyluracil in acetonitrile at time delays from 0 to 600 fs (a) and from 0.60 to 20 ps (b). The excitation wavelength was set at 270 nm. Time zero was set at the maximum amplitude of the stimulated emission band of 1-cyclohexyluracil around 325 nm. Arrows are included to highlight the primary changes in the absorption bands. 125x189mm (600 x 600 DPI)


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Figure 3. (a) Kinetic decay traces of 1-cyclohexyluracil in acetonitrile at selected probe wavelengths. The excitation wavelength was set at 270 nm. (b) Decay associated spectra obtained using a target analysis method, in which a sum of four exponential components plus a constant offset were used to globally-fit the broadband data across the 3 ns time window. As highlighted in the legend, the DAS show some residual contribution from the absorption spectra of other transient species, which is due primarily to the strong overlap of the transient absorption species in the spectral probe windows used (see SI for details). 125x189mm (600 x 600 DPI)



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Figure 4. Singlet and triplet vertical excitation energies for 1-cyclohexyluracil in vacuum and in acetonitrile calculated at the TD-PBE0/6-311++G(d,p)//B3LYP/6-311++G(d,p) level of theory. The IEF-PCM solvation model was used for the vertical excitation energies in acetonitrile (see Methods section in the SI for details). 62x47mm (600 x 600 DPI)


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TABLE OF CONTENTS IMAGE 50x50mm (600 x 600 DPI)


Direct Observation of Triplet-State Population Dynamics in the RNA

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