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J. Phys. Chem. B 2009, 113, 14336–14342
Initial Excited-State Structural Dynamics of Uracil from Resonance Raman Spectroscopy Are Different from Those of Thymine (5-Methyluracil) Soujanya Yarasi, Susan Ng, and Glen R. Loppnow* Department of Chemistry, UniVersity of Alberta, Edmonton, Alberta T6G 2G2, Canada ReceiVed: June 6, 2009; ReVised Manuscript ReceiVed: August 31, 2009
To explore the origin of the differences in UV photochemistry of uracil (RNA) and thymine (DNA) nucleobases, we have measured the UV resonance Raman spectra of uracil in aqueous solution at wavelengths throughout the lowest-energy absorption band and analyzed the resulting resonance Raman excitation profiles and absorption spectra using a time-dependent wave-packet formalism to obtain the initial excited-state structural changes. In contrast to thymine, which differs from uracil only by the presence of a methyl group at C5, most of the resonance Raman intensity and resulting initial excited-state structural dynamics for uracil occur along in-plane hydrogen-bond angle deformation, ring stretching, and carbonyl vibrational modes. Weaker intensities and less significant structural dynamics are observed along the CdC stretching mode. These results suggest that the initial excited-state structural dynamics of uracil occur along a carbon pyramidalization coordinate. These dynamics are different from those of thymine, which distorts primarily along a C5dC6 bond lengthening coordinate. These differences in initial excited-state structural dynamics can explain the different primary photoproducts observed for these two pyrimidine nucleobases. SCHEME 1
Introduction DNA and RNA are responsible for the genetic coding of life and are composed of two purines, adenine and guanine, and three pyrimidines, cytosine, thymine, and uracil.1 Thymine is found only in DNA, and uracil is found only in RNA. The only difference between uracil and thymine (Scheme 1) is the methyl group at position C5 in thymine, whereas uracil has a hydrogen at this position. Thymine and uracil are therefore expected to have similar chemical and photochemical properties. However, it is well-known that, upon UV irradiation to the allowed1(ππ*) state, the thymine (TpT) and uracil (UpU) dinucleotides form different photoproducts.2,3 Thymine dinucleotides preferentially form the cyclobutyl photodimer (CPD) with a quantum yield, φ, of 0.013 as a result of [2 + 2] cycloaddition between the CdC bonds of adjacent thymines or the pyrimidine-pyrimidinone [6-4] photoproduct (φ ) 0.003) as a result of [2 + 2] cycloaddition between the CdC bond of one thymine and the CdO bond of the adjacent thymine.3 Uracil dinucleotides form the photohydrate (φ ) 0.018) as the major photoproduct and the CPD (φ ) 0.007) as the minor photoproduct.4 Whereas DNA is known to have repair mechanisms for photodimers and other damage,5 only one repair mechanism has been found for RNA thus far.6 These results suggest that the structural difference of a single methyl group might be much more important than previously thought. To better understand the origin of these differences in the photochemistry of thymine and uracil, a probe of the initial excited-state structural dynamics is crucial. Femtosecond UV multiphoton ionization has been used to measure gas-phase excited-state lifetimes of thymine and uracil of 6.4 and 2.4 ps, respectively.7 Time-resolved absorption and fluorescence indicate much shorter lifetimes of 0.3-0.7 and 0.5-1 ps for aqueous thymidine and thymidine monophosphate, respectively.8-10 However, these rates represent electronic relaxation only, with * To whom correspondence should be addressed. Phone: 780-492-9704. Fax: 780-492-8231. E-mail:
[email protected].
little information about molecular structural changes occurring in the photochemically active excited state. A recent ultrafast IR probe of the excited-state structural dynamics of (dT)18 suggested that the low CPD quantum yields arise from infrequent conformations that promote the CPD transition state,11 but no information is available regarding the origin of the different UV photochemical products of thymine and uracil. Resonance Raman vibrational scattering spectroscopy provides a probe of initial excited-state structural dynamics.12 Resonant enhancement of those normal vibrational modes coupled to the electronic excitation occurs when the exciting wavelength falls within an absorption band of the system of interest. The intensity of a resonance Raman vibrational band is directly proportional to the slope of the excited-state potential energy surface along that vibrational coordinate; the greater the change in molecular structure in the electronic excited state along a particular nuclear coordinate, the greater the resulting resonance Raman intensity in that vibrational band.12 Resonance Raman studies of several deoxyribonucleotides and ribonucleotides at different excitation wavelengths have been performed.13-15 In the earlier studies, surprisingly, no remarks were made about the differences in relative intensities of vibrational bands for dUMP and dTMP molecules. Only one previous attempt was made to quantify the observed changes in the excited states of these two nucleobases: Peticolas and
10.1021/jp9053378 CCC: $40.75 2009 American Chemical Society Published on Web 09/28/2009
Initial Excited-State Structural Dynamics of Uracil co-workers16 used the Kramers-Kronig transform technique to estimate the amounts of geometry change upon excitation for uracil and thymine. However, the experimental and calculated resonance Raman spectra did not agree. Specifically, the 1235 cm-1 band of uracil was underestimated, and the 1662 cm-1 band of thymine was overestimated with this procedure.16 Recently, a quantitative analysis of the excited-state dynamics of thymine was performed.15 These results showed that thymine initially distorts in the (ππ*) excited state along primarily CdC stretching and in-plane hydrogen-bond angle deformation vibrational modes, with smaller changes along a delocalized ring stretching vibration, which is consistent with the photochemical reaction coordinates for both the CPD and [6-4]photoproduct formation mechanisms. In this work, the initial excited-state dynamics of uracil were determined from the UV resonance Raman spectra excited within its intense, longest-wavelength absorption band centered at 265 nm. The results show the ability of resonance Raman spectroscopy to distinguish the important initial excited-state structural dynamics. Upon excitation, uracil was found to vibrationally relax via primarily CsH deformation modes and modes delocalized over the entire ring. Comparison of the molecular parameters upon photoexcitation with those of thymine15a demonstrates a mode-specific difference in excitedstate structural dynamics between the two pyrimidine nucleobases: uracil evolves toward a more pyramidalized excited-state structure as compared to thymine, which exhibits more of a planar distortion along the C5dC6 double bond.15 These differences in excited-state dynamics upon photoexcitation for the two nucleobases are attributed to normal-mode localization by the methyl group in thymine and are discussed here in the context of the subsequent photochemistry. Experimental Section Uracil (2,4-dioxopyrimidine) (99%, Sigma, Oakville, Ontario, Canada), thymine (5-methyl-2,4-dioxopyrimidine, 99%, Sigma, Oakville, Ontario, Canada), and sodium sulfate (99%, Merck KgaA, Darmstadt, Germany) were obtained commercially and used as supplied. Nanopure water from a Barnstead water filtration system (Boston, MA) was used to prepare the uracil solutions. Sodium sulfate was used as an internal intensity standard and did not have a noticeable effect on either the absorption or resonance Raman spectra of uracil. Typical concentrations of uracil and sulfate for resonance Raman spectroscopy were 3-5 mM and 0.4-0.5 M, respectively. Typical concentrations of thymine and sulfate for resonance Raman spectroscopy were 3-5 mM and 0.4-0.5 M, respectively. The UV resonance Raman laser system, spectrometer, and experimental details for uracil were identical to those described in detail previously.15 Reported frequencies are accurate to (2 cm-1. Measurement of the resonance Raman spectra and determination of the intensities were repeated on three fresh samples of uracil at each wavelength. Analysis of the data was performed as previously described.15 Bleaching of the sample was corrected by averaging the measured solution absorbance before and after each scan. The observed bleaching in a 60min scan was
