Room-Temperature Phosphorescence of the DNA Monomer Analogue

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Room-Temperature Phosphorescence of the DNA Monomer Analogue 4-Thiothymidine in Aqueous Solutions after UVA Excitation Christian Reichardt and Carlos E. Crespo-Hern andez* Center for Chemical Dynamics, Department of Chemistry, Case Western Reserve University, 10900 Euclid Avenue, Cleveland, Ohio 44106

ABSTRACT Room-temperature phosphorescence of the 4-thiothymidine DNA analogue after UVA excitation has been revealed in aqueous buffer solutions at pH 7.4. Femtosecond broad-band transient absorption measurements in combination with quantum chemical calculations that include explicit and implicit solvent effects are used to rationalize this remarkable phenomenon. It is shown that the initial excited singlet-state population bifurcates on the sub-240 fs time scale to populate the triplet state in high yield. A negligibly small fraction of the excited singlet-state population decays by photon emission. SECTION Biophysical Chemistry

T

he excited-state dynamics of DNA and its building blocks have received renewed attention in the past decade thanks to advances in computational methods and ultrafast spectroscopic techniques. Most investigations have focused on understanding the electronic relaxation pathways of DNA in the singlet manifold.1-7 Recent experimental8-11 and computational works5,12-15 have shed some light on the relaxation pathways responsible for the population of the reactive triplet state in several pyrimidine monomers. However, further progress at understanding the role of the long-lived triplet state in DNA photochemistry has been hindered by the intrinsically small triplet quantum yields of the DNA bases and polymers in aqueous solutions.16 We have recently focused our efforts on studying DNA monomer analogues that exhibit high triplet yields to understand how noncovalent interactions modulate their excitedstate dynamics when incorporated into DNA oligonucleotides. An important example is the 4-thiolated deoxyribosylthymine analogue (4-thioThd).17 4-ThioThd can be incorporated into DNA, forming base pairs with the adenine base.18 It has a strong absorption band with a maximum at 335 nm in aqueous solution (Figure 1a), which permits its selective excitation at UVA wavelengths. An increase in the triplet yield of 4-thioThd relative to that of the thymidine (Thd) monomer is expected because the spin-orbit coupling constant of the sulfur atom (386 cm-1) is 2.5-fold higher than that of the oxygen atom (157 cm-1).19 Indeed, a recent study has estimated a triplet quantum yield of unity for 4-thioThd in acetonitrile solutions,20 while a triplet yield of 7 10-2 has been reported for Thd in the same solvent.21 In this Letter, we show that room-temperature phosphorescence can be detected in aqueous buffer solutions of 4-thioThd after UVA excitation. Femtosecond broad-band transient absorption measurements and quantum chemical

r 2010 American Chemical Society

calculations were performed to unravel the relaxation pathways responsible for the efficient population of the triplet state. The experimental setup and computational methods have been presented in detail elsewhere.22,23 Additional experimental and computational details are presented in the Supporting Information (SI). Figure 1a shows the absorption and emission spectra of 4-thioThd in phosphate buffer solution at pH 7.4. The absorption spectrum extends to ∼370 nm, with small residual absorption extending up to ∼400 nm. Strikingly, the emission spectrum shows dual emission bands with maxima at ∼400 and 542 nm. The intensity of the high-energy emission band does not change in presence or absence of molecular oxygen, while that in the visible shows a 3-fold increase when the oxygen concentration is minimized by saturating the solution with N2 gas. The ability of O2 to quench the emission band in the visible suggests that this band should be assigned to phosphorescence, while the unaffected band at around 400 nm can be assigned to fluorescence emission. A zerozero energy transition of 3.43 eV (27 660 cm-1) is estimated for the singlet state from the intersection of the absorption and the emission spectra (Figure 1S, SI).24 The observation of dual emission that originates from two different electronic states is remarkable. We have performed ground- and excited-state calculations using density functional theory with the B3LYP25,26 and the PBE027,28 functionals, respectively, to further support our assignments of the steady-state emission bands on the DNA and RNA monomers of 4-thioThd.24 All calculations were performed using the

Received Date: May 28, 2010 Accepted Date: June 30, 2010 Published on Web Date: July 07, 2010

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DOI: 10.1021/jz100729w |J. Phys. Chem. Lett. 2010, 1, 2239–2243

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We have optimized the T1 state of 4-thioThd at the PBE0/ IEFPCM/6-311þþG(d,p)//UB3LYP/IEFPCM/6-31þG(d,p) level of theory and predicted that phosphorescence should occur at 2.23 eV (557 nm), in agreement with the experimental emission maximum at 542 nm. The reasonably good agreement of the computational results for the T1 state and the observation of O2 quenching of the emission band with a maximum at 542 nm allow us to confidently assign this band to phosphorescence emission from the T1 state. The highenergy emission band with a maximum at around 400 nm can be assigned to fluorescence emission from either the S1 or S2 state. This is because the calculations predict that the S1 state also has significant ππ* character in solution. Fluorescence up-conversion experiments and geometry optimization of the S1 and S2 states are needed to unequivocally assign the fluorescent state. Regardless of which is the fluorescent state, the very low fluorescence intensity of 4-thioThd in aqueous solutions is explained by the extremely fast intersystem crossing (ISC) lifetime and close to unity triplet yield (see below). These assignments are in satisfactory agreement with the calculations taking into consideration the expected accuracy of the level of theory used.32 Femtosecond transient absorption experiments have been performed in the spectral range from 350 to 650 nm to further rationalize the observed dual emission in the emission spectrum of 4-thioThd. Excitation wavelengths of 340 and 360 nm were chosen to investigate how excess vibrational energy in the singlet manifold affects the excited-state dynamics in 4-thioThd. Two exponential terms and a constant offset are needed to adequately fit the spectral evolution in the time window from femtoseconds to 3 ns. The lifetimes obtained from a global fit analysis are 0.24 ( 0.02 and 84 ( 2 ps (errors are reported as 2σ).24 A representative contour plot and transient absorption spectra are shown in Figures 1b and 2, while representative kinetic traces are shown in Figure 3. Importantly, identical excited-state dynamics are observed after exciting 4-thioThd at 340 or 360 nm (not shown). This observation and the lack of excitation wavelength dependence of the steady-state phosphorescence emission band from ∼300 to 360 nm show that excess vibrational energy in the singlet manifold does not modulate the excited-state dynamics of 4-thioThd significantly. The changes in the transient absorption spectra are shown in Figure 2. At delay times shorter than 170 fs, the transient absorption spectra show at least three transient absorption bands (Figure 2a). Two bands below 520 nm show negative signals (ΔA),while the band above 520 nm shows positive signals. An isosbestic point seems to be present at ∼520 nm. The band below 370 nm results primarily from ground-state depopulation, in agreement with the absorption spectrum reported in Figure 1a. The band with a negative ΔA maximum at ∼420 nm is assigned to stimulated emission. Evidence for this assignment comes from the weak fluorescence emission band shown in the emission spectrum, Figure 1a. The stimulated emission signal decays with a lifetime of 0.24 ps, while a simultaneous increase in absorption above 520 nm is observed, Figures 2b and 3. This transient absorption band, which has maximum at ∼560 nm (Figure 2c), strongly resembles the triplet-triplet absorption band

Figure 1. (a) Steady-state absorption (black) and emission spectra of 4-thioThd at room temperature after excitation at 310 nm in N2(red) and air-saturated (blue) solutions. (b) Contour plot after excitation at 340 nm in aqueous buffer solution at pH 7.4.

Gaussian03 suite of programs.29 Bulk (IEFPCM)30,31 and explicit solvent effects have been taken into consideration in the ground- and excited-state calculations.24 The vertical excitation energies of the first five excited states of 4-thioThd and of the 4-thioThd(H2O)2 complex were predicted at the PBE0/IEFPCM/ 6-311þþG(d,p)//B3LYP/6-311þþG(d,p) and PBE0/IEFPCM/6311þþG(d,p)//B3LYP/6-31þG(d,p) levels of theory, respectively (Table 1S, SI). The character of the excited states was determined from the effects that bulk and explicit water solvent have on the excitation energies and from a molecular orbital analysis of the principal configuration interaction transitions. The most relevant molecular orbitals are shown in Figure 3S (SI).24 The lowest-energy singlet and triplet states of 4-thioThd and of the 4-thioThd(H2O)2 complex in bulk water solvent (percentages for the complex are in parentheses) have approximately 18% (37%) ππ* and 82% (63%) nπ* character for the S1 state and 84% (64%) ππ* and 16% (36%) nπ* character for the T1 state.24 The S2 state has 100% ππ* character in both the 4-thioThd and the 4-thioThd(H2O)2 complex in solution. The vertical excitation energies for the 4-thioThd(H2O)2 complex are predicted to be 3.22 (385 nm), 2.59 (478 nm), and 4.00 eV (310 nm) for the S1, T1, and S2 states, respectively, in good agreement with the experimental results shown in Figure 1a. The vertical excitation energies for 4-thioThd are predicted at 3.26 (380 nm), 2.60 (477 nm), and 4.02 eV (308 nm) for S1, T1, and S2 states, respectively. Analogous calculations in the gas phase for 4-thioThd predict 2.99, 2.38, and 4.13 eV vertical energies for the S1, T1, and S2 states, respectively.24


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Scheme 1. Proposed Kinetic Mechanism Explaining the Dual Emission Bands Observed in 4-thioThd in Aqueous Buffer Solutions after Excitation at 340 or 360 nm at Room Temperaturea

a

The bulk of the initial excited-state population in the S2 state decays by ultrafast intersystem crossing to the triplet state. Only a negligibly small fluorescence is observed while the triplet-state population decays by phosphorescence and other nonradiative quenching processes. Thin grey lines represent alternative relaxation pathways (see text for discussion).

the visible (Figure 2a,b), which it is likely due to the contribution of the stimulated emission signal at probe wavelengths below ∼550 nm at those time delays. We consider ultrafast internal conversion from the S2 to S1 state8-10,14,15 as an alternative explanation for the apparent blue shift of this band, but a global fit analysis using three exponential functions does not support the need for the additional component. A fraction (∼15%) of the triplet-state population decays back to the ground state with a lifetime of ∼80 ps (Figures 2c and 3). We tentatively assign this process to partial quenching of the triplet state by either triplet-triplet annihilation or triplet-self-quenching relaxation pathway. Evidence in support of this hypothesis comes from the observation of partial ground-state recovery with an identical lifetime (Figure 3) and from the effect that the viscosity of a given solvent has on the lifetime associated with this relaxation pathway.17 The partial recovery of the ground state seems to rule out product formation, solvent relaxation, or vibrational cooling dynamics in the triplet manifold as alternative relaxation pathways. However, the 80-ps lifetime is difficult to reconcile with a bimolecular quenching pathway taking into consideration the concentration of 4-thioThd used (0.4 mM). An explanation for this apparent discrepancy is that a small fraction of the 4-thioThd molecules is present in a preorganized state (i.e., a loose ground-state complex or aggregate state), such that diffusional encounter is no longer required. Unfortunately, to our knowledge, the association constant of 4-thioThd in aqueous buffer solutions is unknown. Further experiments are needed in which the concentration of 4-thioThd or the temperature of the solution is systematically varied. In a nutshell, the simplest kinetic model that can satisfactorily explain our experimental and computational observations is shown in Scheme 1. Excitation of 4-thioThd by UVA light results in ultrafast branching of the initial S2 state population. A negligibly small fraction of the S2 state decays back to the ground state by photon emission. No evidence of ultrafast internal conversion to the ground state was obtained. On the contrary, most of the initial excited-state population decays through an essentially barrierless path that connects the S2 state to the T1 state, resulting in an ISC rate constant of ∼4.2 1012 s-1 and a triplet yield close to unity. This suggests

Figure 2. Transient absorption spectra of 4-thioThd in pH 7.4 phosphate buffer solution after excitation at 340 nm: (a) early dynamics; (b) delay times from 170 fs to 2 ps; and (c) delay times from 2 to 460 ps. Water stimulated Raman emission is observed at 385 nm at short time delays.

Figure 3. Representative transient absorption signals (circles) at the specified probe wavelengths. Best global-fit curves are shown by solid lines (see SI for details and additional probe wavelengths, Figure 4S).

measured recently after 263 nm excitation of 4-thioThd in aqueous buffer solution using transient absorption spectroscopy.33 Thus, we assign this band to the T1 state.17 We also estimate a triplet quantum yield close to unity from backto-back experiments in acetonitrile and aqueous buffer solutions under identical conditions24 using the triplet yield recently reported for 4-thioThd in acetonitrile.20 An apparent blue-shift is observed for the triplet-triplet absorption band in


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that a conical intersection may lie along the ISC decay pathway, as recent calculations have proposed for uracil and thymine.14,34 Note that we cannot rule out unequivocally the participation of the S1 state in the excited-state dynamics of 4-thioThd. If the S1 state is involved, a mechanism analogous to that reported for the natural pyrimidine monomers is expected.8,9,15 High-level quantum-dynamical calculations and up-conversion experiments are needed to achieve a complete picture of the competing relaxation pathways. Significantly, a fraction of the triplet-state population decays back to the ground state by phosphorescence emission while another small fraction is quenched on the picosecond time scale. We anticipate that yet another fraction of the triplet-state population is quenched at longer time scales because the high triplet yield is not fully accounted for by the relatively low yield of phosphorescence and the picosecond quenching processes. Additionally, a small fraction of the excited-state population undergoes photochemistry, as revealed by comparing the steady-state absorption spectra of 4-thioThd before and after the laser experiments.17 The kinetic model presented here might be compared with that proposed very recently by Harada et al. for the case of excitation at 263 nm.33 The authors proposed that ISC to the triplet state occurs in less than 10 ps and in high yield, in qualitative agreement with our results. However, they were unable to measure the ISC rate constant or stimulated emission probably because of the limited time resolution of their experimental setup. In addition, the authors observed a sub10 ps relaxation pathway proposed to result from either vibrational cooling in the T1 state or slow internal conversion from the S2 to the S1 state when probing at 500 and 570 nm.33 In our experiments, direct excitation of the S2 state with either 340 or 360 nm did not provide evidence that excess vibrational energy in the singlet manifold results in significant vibrational cooling in the triplet state or slow S1 r S2 internal conversion. It is possible, however, that excitation of 4-thioThd at 263 nm might open other relaxation pathways not accessed when exciting at 340 or 360 nm, which could explain their observation. The experimental results presented herein can be used to benchmark recent theoretical efforts at understanding the role of the triplet state in DNA photochemistry.5,12-15,34 The remarkably fast ISC lifetime and the high triplet yield makes 4-thioThd an ideal model base to test the accuracy and predictions of current computational methods.

Supercomputer Center for generous allotment of computer time. C.R. thanks the Deutsche Forschungsgemeinschaft (DFG) for support.

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SUPPORTING INFORMATION AVAILABLE Experimental

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and computational details as well as supporting results. This material is available free of charge via the Internet at http://pubs.acs.org. (14)

AUTHOR INFORMATION Corresponding Author:

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*To whom correspondence should be addressed. E-mail: carlos. [email protected].

ACKNOWLEDGMENT The authors thank the Department of

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Chemistry, Case Western Reserve University for support and the Mississippi Center for Supercomputer Research and the Ohio


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Crespo-Hern andez, C. E.; Cohen, B.; Hare, P. M.; Kohler, B. Ultrafast Excited-State Dynamics in Nucleic Acids. Chem. Rev. 2004, 104, 1977–2019. de Vries, M. S.; Hobza, P. Gas-Phase Spectroscopy of Biomolecular Building Blocks. Annu. Rev. Phys. Chem. 2007, 58, 585–612. Markovitsi, D.; Gustavsson, T.; Talbot, F. Excited States and Energy Transfer among DNA Bases in Double Helices. Photochem. Photobiol. Sci. 2007, 6, 717–724. Middleton, C. T.; de La Harpe, K.; Su, C.; Law, Y. K.; CrespoHern andez, C. E.; Kohler, B. DNA Excited-State Dynamics: From Single Bases to the Double Helix. Annu. Rev. Phys. Chem. 2009, 60, 217–239. Serrano-Andr es, L.; Merch an, 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. Towrie, M.; Doorley, G. W.; George, M. W.; Parker, A. W.; Quinn, S. J.; Kelly, J. M. ps-TRIR Covers All the Bases ; Recent Advances in the Use of Transient Ir for the Detection of ShortLived Species in Nucleic Acids. Analyst 2009, 134, 1265–1273. Zgierski, M. Z.; Fujiwara, T.; Lim, E. C. Conical Intersections and Ultrafast Intramolecular Excited-State Dynamics in Nucleic Acid Bases and Electron Donor-Acceptor Molecules. Chem. Phys. Lett. 2008, 463, 289–299. Hare, P. M.; Crespo-Hern andez, C. E.; Kohler, B. SolventDependent Photophysics of 1-Cyclohexyluracil: Ultrafast Branching in the Initial Bright State Leads Nonradiatively to the Electronic Ground State and a Long-Lived 1nπ* State. J. Phys. Chem. B 2006, 110, 18641–18650. Hare, P. M.; Crespo-Hern andez, C. E.; Kohler, B. Internal Conversion to the Electronic Ground State Occurs via Two Distinct Pathways for Pyrimidine Bases in Aqueous Solution. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 435–440. Kwok, W.-M.; Ma, C.; Phillips, D. L. A Doorway State Leads to Photostability or Triplet Photodamage in Thymine DNA. J. Am. Chem. Soc. 2008, 130, 5131–5139. Hare, P. M.; Middleton, C. T.; Mertel, K. I.; Herbert, J. M.; Kohler, B. Time-Resolved Infrared Spectroscopy of the Lowest Triplet State of Thymine and Thymidine. Chem. Phys. 2008, 347, 383–392. Marian, C. M.; Schneider, F.; Kleinschmidt, M.; Tatchen, J. Electronic Excitation and Singlet-Triplet Coupling in Uracil Tautomers and Uracil-Water Complexes a Quantum Chemical Investigation. Eur. Phys. J. D 2002, 20, 357–367. Merch an, M.; Serrano-Andr es, L.; Robb, M. A.; Blancafort, L. Triplet-State Formation along the Ultrafast Decay of Excited Singlet Cytosine. J. Am. Chem. Soc. 2005, 127, 1820–1825. Serrano-Andr es, J. J.; Gonz alez-Luque, R.; Merch an, M.; Serrano-Andr es, L. On the Intrinsic Population of the Lowest Triplet State of Thymine. J. Phys. Chem. B 2007, 111, 11880– 11883. Etinski, M.; Fleig, T.; Mirian, C. M. Intersystem Crossing and Characterization of Dark States in the Pyrimidine Nucleobases Uracil, Thymine, And 1-Methylthymine. J. Phys. Chem. A. 2009, 113, 11809–11816. Cadet, J.; Vigny, P. The Photochemistry of Nucleic Acids. In Bioorganic Photochemistry; Morrison, H., Ed.; Wiley-Interscience: New York, 1990; Vol. 1, pp 1-272.


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Reichardt, C.; Crespo-Hern andez, C. E. Ultrafast Spin Crossover in 4-Thiothymidine in an Ionic Liquid. Chem. Commun. 2010, DOI: 10.1039/c0cc01181a. Massey, A.; Xu, Y.-Z.; Karran, P. Photoactivation of DNA Thiobases As a Potential Novel Therapeutic Option. Curr. Biol. 2001, 11, 142–1146. Schoonheydt, R. A.; Lunsford, J. H. An Electron Paramagnetic Resonance Study of SO2- on Magnesium Oxide. J. Phys. Chem. 1972, 76, 323–328. Harada, Y.; Suzuki, T.; Ichimura, T.; Xu, Y.-Z. Triplet Formation of 4-Thiothimidine and Its Photosensitization to Oxygen Studied by Time-Resolved Thermal Lensing Technique. J. Phys. Chem. B 2007, 111, 5518–5524. Salet, C.; Bensasson, R.; Becker, R. S. Triplet Excited States of Pyrimidine Nucleosides and Nucleotides. Photochem. Photobiol. 1979, 30, 325–329. Reichardt, C.; Vogt, R. A.; Crespo-Hern andez, C. E. On the Origin of Ultrafast Nonradiative Transitions in NitroPolycyclic Aromatic Hydrocarbons: Excited-State Dynamics in 1-Nitronaphthalene. J. Chem. Phys. 2009, 131, 224518. Vogt, R. A.; Peay, M. A.; Gray, T. G.; Crespo-Hern andez, C. E. Excited-State Dynamics of (Organophosphine)gold(I) Pyrenyl Isomers. J. Phys. Chem. Lett. 2010, 1, 1205–1211. See Supporting Information for details. Lee, C.; Yang, W.; Parr, R. G. Development of the ColleSalvetti Correlation-Energy Formula into a Functional of the Electron Density. Phys. Rev. 1988, B37, 785–789. Becke, A. D. A New Mixing of Hartree-Fock and Local Density-Functional Theories. J. Chem. Phys. 1993, 98, 1372–1377. Adamo, C.; Barone, V. Toward Reliable Density Functional Methods without Adjustable Parameters: The PBE0 Model. J. Chem. Phys. 1999, 110, 6158–6170. Adamo, C.; Scuseria, G. E.; Barone, V. Accurate Excitation Energies from Time-Dependent Density Functional Theory: Assessing the PBE0 Model. J. Chem. Phys. 1999, 111, 2889– 2899. Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Zakrzewski, V. G.; Montgomery Jr., J. A.; Stratmann, R. E.; Burant, J. C.; et al. Gaussian 03, revision D.01; Gaussian, Inc.: Wallingford, CT, 2004. Barone, V.; Cossi, M.; Tomasi, J. A New Definition of Cavities for the Computation of Solvation Free Energies by Polarizable Continuum Model. J. Chem. Phys. 1997, 107, 3210–3221. Cances, E.; Mennucci, B.; Tomasi, J. A New Integral Equation Formalism for the Polarizable Continuum Model: Theoretical Background and Applications to Isotropic and Anisotropic Dielectrics. J. Chem. Phys. 1997, 107, 3032–3041. Jacquemin, D.; Wathelet, V.; Perp ete, E. A.; Adamo, C. Extensive TD-DFT Benchmark: Singlet-Excited States of Organic Molecules. J. Chem. Theory Compt. 2009, 5, 2420–2435. Harada, Y.; Okabe, C.; Kobayashi, T.; Suzuki, T.; Ichimura, T.; Nishi, N.; Xu, Y.-Z. Ultrafast Intersystem Crossing of 4-Thiothymidine in Aqueous Solution. J. Phys. Chem. Lett. 2010, 1, 480–484. Climent, T.; Gonz alez-Luque, R.; Merch an, M.; SerranoAndres, L. On the Intrinsic Population of the Lowest Triplet State of Uracil. Chem. Phys. Lett. 2007, 441, 327–331.

NOTE ADDED AFTER ASAP PUBLICATION This Letter published ASAP on July 7, 2010. Reference 17 was updated with the correct DOI. The correct version was published on July 16, 2010.


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Room-Temperature Phosphorescence of the DNA Monomer Analogue

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