J . Phys. Chem. 1993,97, 13515-13518
13515
Electronic Spectra of the Neutral Radical and H and OH Adducts of Uracil M. Krauss’ Center for Advanced Research in Biotechnology, National Institute of Standards and Technology, 9600 Gudelsky Drive, Rockville. Maryland 20850
R. Osman Department of Physiology and Biophysics, Mount Sinai School of Medicine, City University of New York, New York, New York 10029 Received: July 16,1993; In Final Form: October 4, 1993”
Theoretical assignments of the radicals of nucleic acid bases and the radical adducts are compared with experiment for the simplest base, uracil. The electronic excitation energies of H and OH adducts of uracil are calculated by ab initio methods to provide a catalogue of the electronic states and transitions for such radicals. The C5 and C6 adducts for both H and OH are found to support spectral transitions in entirely different regions. This result contradicts an experimental deduction that the C5 and C6 H adduct spectra have a similar shape. Continuum solvation reaction field shifts from the in vacuo transition energies are substantial and important in assigning transitions. The neutral radical spectrum is also analyzed since the addition of OH is suggested experimentally to result ultimately in the spectrum of this radical. Theoretical transitions are found to span the observed spectral region. Earlier studies of aromatic hydrocarbon radicals support the accuracy of the calculations. An additional test was made here by calculating the phosphorescent transition for the triplet state of uracil.
Introduction The radicals created in radiation damageof nucleic acids initiate chemical reactions that result in strand damage. These radicals arise from the primary ionization of the nucleic acid or from reactions with the hydrated electron and radicals that are formed in aqueous solution.’ Understanding the overall mechanism requires an accounting of the time dependence of radical formation. Identification of these radical species is generally attempted with both electron spin resonance and transient absorption spectra. However, little is known of the electronic properties of the radicals and the relative stabilities of isomers. Although there is an extensive literature on the spectroscopy of the individual bases, nucleosides, and nucleotides? the radicals are not well known and the assignments can be ambiguous. In this article we will focus on the spectroscopy of the adducts of H and OH to uracil to show that experimental assignments are difficult even when a model system of a single base is examined. Addition of radicals at the C 5 4 6 bond of uracil leads to two isomers with the radical bonded to either the C5 or C6 atom. The standard numbering scheme for uracil is given in Figure l a together with a schematic representation of the 5-yl radical that results from addition of OH to the C6 atom. The different electronic and chemical behavior of these two adducts can be examined in the model case of the base, uracil. The spectrum of both 5H (6-yl radical) and 6H (5-yl radical) adducts has been deduced for uraciL3 Very different redox properties have been determined for C5 and C6 adducts of H and OH. The electronic properties of C5 and C6 adducts would then be different and necessarilylead to different spectra. This differencein electronic properties will be shown below to arise from the extent of coupling of the open-shell orbital into the r orbital of the carbonyl bonds. However, the experimentalspectra of the H adducts are deduced to be similar except for intensities, Differences in peak maxima for the tautomers are noted for transient spectra resulting from the addition of OH to uracil, but transitions in the visible are expected for both tautomers. Most experimentsyield the spectra of both the C5 and C6 OH adductsbut are predominatelyassigned Abstract published in Aduance ACS Abstracts, December 1, 1993.
0022-3654/93/2097-13515$04.00/0
to the 6-yl radicals.”8 For example, the reaction of the C5OH(6-yl) uracil radical is followed at 400 nm.5 The 5-yl spectrum of the OH adduct to uracil is obtained by electron-transfer dissociation of bromine from 5-bromo-5,6-dihydro-6-hydroxy~yrimidine.~ At high pH, the spectrum observed subsequent to the addition of OH to uracil is assigned to the neutral uracil radical and its conjugate base anion.8 Theoretical electronic assignments of DNA base radicals are almost totally lacking except for the triplet absorptionsfor which there are extensive calculations.2J0J The ground-stateelectronic and geometric structure of the DNA base radicals has been studied,12-14but there is little or no ab initio information on the excited states. Recently, we have shown that a first-order configuration interaction (FOCI) relative to a multireference base set of configurations can provide moderately accurate excitation energies for radicals and anions15and is feasible for valence states of systems as large as indole.16 The accuracy of the representation of the relevant valence states depends on the extent to which the active valence orbitals models the bond breaking that accompanies the excitation. The valence states are dominated either by transferring the hole in the dominant Hartree-Fock (HF) representation of the ground state among H F occupied orbitals or by the excitation to the lowest unoccupied orbital that correlates the bond or bonds being broken. It is assumed that the remaining dynamical correlations in the bonds are similar in these valence states and cancel. FOCI calculationsof the excitation energies of the C5 and C6 adducts of uracil with H and OH will allow a comparison with the experimentaldeductionsregarding the comparativebehavior of the spectra of theseradicals. The neutral radical can be formed subsequent to OH addition by hydrolysis, and the spectrum is alsocalculatedto assist the analysis. The significant disagreement found with experimental assignments for both H and OH tautomer adducts shows the need for more theoretical input into the assignment of radical spectra. Method The FOCI multiconfigurationreference state among theactive orbitals provides all possible couplings equivalent to a complete 0 1993 American Chemical Society
Krauss and a m a n
13516 The Journal of Physical Chemistry, Vol. 97, No. 51, 1993
a
TABLE l: Uracil Radical and Adduct Excitation Energies
0
.ad Moments
final state (nm, fim,f") state 1
0
N O H H
1
H 6H Adduct
C
5.29
443.8 2.99 0.003
4.62
250.2 7.18 0.005
5.53
416.8 2.91
4.94
243.1 8.14
5.34
3.83
_-f-:
2.86
0 A
61.1.
t
3.52
d
5H Adduct
0
s.,.
I
St.!.
2
Figure 1. (a) Schematic of uracil and atom numbex designation. (b) C60H (5-yl) adduct of uracil. (c, d) Schematicof magnitude and phase of the atomic function coefficients in the open-shell natural orbital (Le., occupation number close to 1) for (c) C6H adduct and (d) C5H adduct.
active space (CAS) reference. Single excitations from this reference allows for relaxation of the initially chosen molecular orbital basis. Natural orbitals are extracted from the configuration interaction (CI) solutions and iterated" to improve the convergenceof the CI. The iteration optimizesthe valenceorbitals for the CI. The dominant active orbitals are defined as the valence orbitals for both ground and excited states. These orbitals must be of comparable radial size, ruling out the calculation of valence and Rydberg states within the same calculation. This procedure ensures that all excited states are catalogued within a specified energy range that avoidsthe Rydberg states. However, excitations from tightly bound orbitals are restricted because of limitations in the size of the configuration interaction matrix that can be accommodated. Selective relaxation of the restrictions are examined to determine whether the excitation energies for the chosen active space do not change substantially. The limiting behavior is estimated by increasing the number of active orbitals as the natural orbital basis is truncated when the occupany falls below 1 V during iterations. Only one additional active orbital was used with the occupied orbitals of the restricted open-shell HartreeFock configuration. The occupation number of this natural orbital always exceeded that of the next NO by at least four, and those orbitals had coefficients below 0.05. Tests of these procedures have been made to the excitation energies of the phenyl,'* benzyl, phenol cation, and phenoxyli9 radicals. Cal-
2
3
4
5
6
6H adduct 341.8 2.12 0.021 5H adduct 234.3 3.02 O.Oo0
6H 1-methyladduct 322.2 3.24 5H 1-methyladduct
60H adduct 341.4 3.60 0.024 SOH axial adduct 240.0 238.5 6.17 2.56 SOH equatorial adduct 246.9 224.1 6.15 4.28 uracil neutral radical (-HN1) 582.6 526.1 461.9 424.0 4.18 2.14 2.05 0.57 0.001 0.018 0.024 0.015 411.6 2.86 0.002
321.2 2.61 0.003
"fis the oscillator strength. culated benzyl excitation energies of 448 and 441 nm to the nearly degenerate excited states compare well with the adiabatic experimental excitation energy of 455 nm to the mixed electronic state.z0 For the phenoxy1 radical a strong transition is predicted at 385 nm, which is to be compared to the well-known transition at 398 nm,zi Energy gradient optimized ground-state structures are determined at theself-consistent-field (SCF) level for all model systems which include the following: -H1 and-H3 neutral radicals; lowest triplet, T1; four H adducts to C5, C6,02,04; two OH adducts to C5, C6; two H adducts to C5 and C6 of I-methyluracil. Both unrestricted Hartree-Fock (UHF) and restricted open-shell HartrebFock (ROHF) calculations were used. Details of the calculation and examination of the electronic structure will be given elsewhere,together with a more comprehensivelist of uracilderived radicals. Since the FOCI calculations could not be done with polarized basis sets at this time, ground-state geometries were determined with basis sets comparable to those used for the FOCI. All FOCI calculations used the CEP-31G basis for only the valence electrons and their concomitant effective core potentialszz to replace the K-shell electron cores. Both UHF geometry optimizations and energetics at the MP2 level were done with GAUSSIANz3using the 6-31G basis. All the FOCI calculations were done with GAMESS.z4 The FOCI energies, dipole moments, and transition moments of the C5 and C6 H and OH adducts to uracil as well as the neutral radical are given in Table 1. Transition moments used the FOCI wave functions and were calculated by averaging the dipole and velocity components. The structures of the systems relevant to the spectroscopyare given in the supplementarytables. Absorption shifts are estimated from classical formula for the dipolbdipole interaction between the solute and Assumingthe solvent dipoles do not rearrange during the electronic transition and the reaction field is frozen but the i n d u d dipoles of the solvent relax, the classical shift in the excitation energy is
The Journal of Physical Chemistry, Vol. 97, No. 51, 1993 13517
Electronic Spectra of Uracil Radicals given by
+
wheref(D) = (D - 1)/(2D + 1) and g(n) = (n2 - 1)/2n2 1) with D the static dielectric constant and n the refractive index of water. Quantum shifts have been calculated from the dipole reaction field incorporated in the Hamiltonian for FOCI calculationsof the indole radical.16 The quantum and classical shifts are not equivalent, but comparable magnitudes were calculated for indole. Only the classical estimates of the shift are reported here for the dipoledipole interaction. Additional red shifts would result from interactions involving the polarizability of the solvent and dispersion. This suggeststhat the sizable blue shifts reported below are overestimates. The lowest triplet valence state of uracil is also reported here to provide support for the accuracy of the calculations. Although tests were made earlier for the indoleradical and radical derivatives of benzene, the uracil radicals are essentially more complicated electronically because of the heteroatoms in the ring and the presence of carbonyl chromophores. At the calculated geometry of the triplet state of uracil, the phosphorescence energy of the lowest triplet energy to the ground singlet energy was calculated with comparable FOCI wave functions to those used for the radicals considered above. A vertical excitationenergy of 22 440 cm-I compares well with the lower bound for the experimental phosphorescence energy of 22 000 cm-1.2,26
Results and Discussion Hydrogen adducts are found across the C 5 4 6 double bond
or on the carbonyl oxygen. Protonation of the electron adduct of uracil has been experimentally deduced to occur first at 0 4 ( 4 4 ) and then to convert slowly to the more stable C6 a d d u ~ t . ~ . ~This ’ , ~experiment ~ provides spectra attributed to both types of a d d ~ c t s . The ~ ~ ?oxygen ~ ~ protonated adduct is observed at low pH, and the assigned spectrum peaks around 300 nm. At later times under suitable pH and buffer conditions a broad spectrum peaked around 405 nm attributed to protonation at carbon is observed. This spectrum was deduced to be primarily due to the 5-yl radical formed by protonation at C6.3*28 The theoretical predictions of the spectra of the H adducts to CS and C6 are very dissimilar. The HC5 adduct has transitions to the blue of 250 nm while the HC6 adduct exhibits both a visible and a near-UV transition in addition to transitions in the far-UV. The theoretical spectra can be compared immediately to experimental spectra for the H One peak is noted experimentally with a maximum around 400 nm for uracil and another around 330 nm. The coalesced peaks are quite broad and extend from 300 to almost 500 nm. However, the spectrum observed by OH radical attack on dihydrouracil is substantially different at shorter wavelengths with a rapidly rising absorption around 300 nm. Even in this case the 6-yl radical is found to be d ~ m i n a n t ?and ~ the spectrum is assigned to this tautomer. The calculated in vacuo peak of 444 nm for the 5-yl radical will be shifted appreciably to the blue in aqueous solution since the dipole moment of the excited state is about 2 D smaller than the ground state. Using the classical f o r m ~ l a t i o n we , ~ ~obtain approximately 397 nm. A peak at 342 nm is also calculated in vacuowhich shifts appreciablyin water to 305 nm. The predicted transitions areroughly in accord with theOH attacke~periment?~ including the relative transition oscillator strengths, The calculation predicts substantially different relative intensities from the experiment which adds the H atom directly. Optimized geometries and energies at the ROHF level were determined for the H adducts to both carbonyl oxygens. The H 0 4 adduct is about 154 W/mol more stable in vacuo than the H 0 2 but is itself about 75 kJ/mol less stable than the C6 adduct at the FOCI level. A transition is calculated for H 0 4 in vacuo at 389 nm and predicted to red-shift to 401 nmin aqueous solution.
The experimental assignment of a peak around 300 nm to H 0 4 is not supported. In fact, the H 0 4 and HC6 transitions are predicted to lie close to one another and be difficult todistinguish. The relative energetics between H 0 4 and the carbon adducts is also troubling considering the experimental suggestion for protonation initially at oxygen. Solvated energeticsmay not find the tautomers as widely separated in energy. The kinetics of proton addition may also becomplicated by the complexity of the anion electronicstructure. An ROHF calculationof the electron adduct anion radical finds the excess spin and open-shell orbital dominated by charge on the C6 atom. However, an FOCI calculation based on the ROHF vectors finds at least four electronic states within 33 kJ/mol of each other. The present basis is not suitable for an in vacuo calculation of the anion, and what is suitable for a solvated anion is still to be determined. Optimized geometriesmay also alter substantially from in vacuo to solvation calculations. This preliminary estimate of the electronic structure of the anion suggests that more than one electronic state plays a role in the protonation, and it is not clear that the apparent pKdifferences between the oxygen and carbon protonation sites represent relative energetics on a ground-state energy surface. Comparable FOCI calculations were done for the C5 and C6 H adducts of l-methyluracil. The transitions for the H C6 adduct shift significantly to the blue from uracil to in vacuo values of 417 and 322 nm. The relative dipole moment differencebetween the ground and excited states again suggests substantial blue shifts to 382 and 288 nm. The experimental spectrum exhibits a broad peak with a definite maximum around 410 nm. In the neighborhood of 425 nm there is either another coalesced peak or curvature in different experiments designed to produce the H adducts to the C 5 4 6 bond.’ Again, there is a rough correspondence to the experiment that shows roughly one peak3above 300 nm. However, the spectra may not be entirely due to the H adducts. Attempts to decompose and assign the spectra further consider the possibility that the adduct to both CS and C6 contributes. There is experimental evidence that the C5 6-yl adduct is produced dominantly,28and substantial spectral absorption is attributed to this adduct. However, the CSH adduct is calculated to have no transitions in the 300-500-nm region at all. The CSH excitation energies in Table 1 are given for the lowest-energyoptimized structure which is nonplanar, but a C5H planar structure was also examined with very similar excitation energies. This conclusion also holds for a planar ring geometry for the H adduct to CS in l-methyluracil. The qualitative difference in the electronic behavior of the C5H and C6H adducts is determined by the relative energy of theopen-shellradical orbitals created in delocalization from either the initial C5 or C6 carbon site. In the C6H adduct the radical site neighbors the carbonyl bond. Two low-lying excited states result from both an in-plane and out-of-plane singly-occupied orbital with the largest component on the 0 4 atom. The natural orbital wave functions for the open-shell orbital in ground and excited states provides insight into the stabilization process. In Figure l b the in-plane open-shell orbital for the C6H excited state is seen to be bonding on the carbonyl. The C5H adduct apparently cannot delocalize toward the N1 atom, which is energetically more costly. The open-shell orbital in this case is a linear combinationof the antibondingcarbonyl orbitals as seen in Figure 1c. The proximity of the initially formed singly occupied orbital localized on C5 to an accessible carbonyl ?r orbital yields the lower excited states of the 6H adduct. The theoretical predictions for the OH adducts to uracil are similar to those for the H adducts. Two CSOH adducts were examined with an axial and equatorial orientationof the hydroxyl group.12 For both structures transitions are found only to the blue of 250 nm. The C60H adduct has in vacuo transitions predicted at 478 and 347 nm which are shifted to 422 and 317
13518 The Journal of Physical Chemistry, Vol. 97, No. 51, 1993
Krauss and &man
cataloguing the spectra of the radicals expected to be present nm in aqueous solution. The absorption spectrum of the C60H which should materially assist the modeling of the kinetics. adduct radical of 1,3-dimethyluraciI has been reported.g This spectrum shows a peak around 360 nm and a long shoulder Supplementary Material Available: Table of internal coordiextending from 425 to 500 nm. The theoretical calculation of nates of the H and OH adducts of uracil in Z-matrix format (3 the C60H adduct roughly corresponds to this spectrum. The pages). Ordering information is given on any current masthead spectrum produced by the reaction of OH with 1,3-dimethyluracil is quite different, and the calculated spectrum attributed to the page. C50H radical has a rapidly rising absorbance to wavelengths References and Notes shorter than 300 nm and a lower intensity but broad peak around 400 nm. Thecalculated spectrum of the C50H adduct contradicts (1) von Sonntag, C. Chemical Basis of Radiation Biology; Taylor and this deduced spectrum. Francis: London, 1987. (2) Callis, P. R. Annu. Rev. Phys. Chem. 1983, 34, 329. Three strong in vacuo transitions are predicted for the neutral (3) Deeble, D. J.; Das, S.; von Sonntag, C. J . Phys. Chem. 1985,89, uracil radical (-HNI) at 526,462, and 424 nm. These transitions 5784. span the broad experimental absorption structure, and a fit could (4) Myers, L. S.; Hollis, M. L.; Theard, L. M.; Peterson, F. C.; Warnick, Chem. SOC.1970, 92, 2875. roughly be made to the spectrum attributed to this r a d i ~ a l . ~ ~A.~ J. ~~ ( 5 ) Hayon, E.; Simic, M. J. Am. Chem. Soc. 1973, 95, 1029. A weaker feature is found further to the red at 583 nm which also (6) Bansal, K. M.; Fessenden, R. W. Radiat. Res. 1978, 75, 497. would contribute to the broad feature found experimentally. An (7) Steenken,S.; Jagannadham, V. J . Am. Chem.Soc. 1985,107,6818. ( 8 ) Fujita, S.;Steenken, S. J. Am. Chem. SOC.1981, 103, 2540. additional in vacuo theoretical peak is predicted to the blue at (9) Deeble, D. J.; von Sonntag, C. 2.Naturforsch. 1985, 40C, 925. 321 nm. It should shift only slightly further to the blue and can (10) Petke, J. D.; Maggiora, G. M.; Christoffersen, R. E. J . Am. Chem. be used to confirm the pattern of the radical absorption. These SOC.1990,112, 5452. results suggest that the calculations are reasonably accurate; no (1 1) Petke, J. D.; Maggiora, G. M.; Christoffersen, R. E. J . Phys. Chem. 1992, 96, 6992. substantial shift in the excitation energies is required for a fit to (12) Osman, R.; Miaskewicz,K.; Weinstein, H. In Physical and Chemical the experiment, but the broadness of the peaks renders comMechanism in Molecular Radiation Biology; Glass, W. A,, Varma, M. N., parisons ambiguous. Eds.; Plenum Press: New York, 1991; pp 423452. (13) Colson, A.; Besler, B.; Close, D. M.; Sevilla, M. D. J. Phys. Chem. The removal of the hydrogen atom from N3 yields an isomer 1992, 96, 661. that is calculated at the in vacuo FOCI level to be only 17 kJ/mol (14) Rashid, R.; Mark, F.; Schuchmann, H. P.; von Sonntag, C. Znt. J . higher in energy than HNI . Since the dipole moment of HN3 Radiat. Biol. 1991, 59, 1081. (15) Krauss, M.; Roszak, S. J. Phys. Chem. 1992,96, 8325. is 7.2 D compared to the smaller value of 3.4 D for H N l , it is (16) Krauss, M.; Garmer, D. R. J. Phys. Chem. 1993, 97, 831. possible that solvation will narrow this gap. Excited states of the (17) Bender, C. F.; Davidson, E. R. J. Phys. Chem. 1966, 70.2675. HN3 isomer are all lower in energy than those for HN1 with the (18) Krauss, M.; Roszak, S. J . Mol. Struct. (THEOCHEM), in press. fourth and fifth predicted to have in vacuo transitions at 448 and (19) Krauss, M. J. Mol. Strucr. (THEOCHEM), in press. (20) Cossart-Magos, C.; Leach, S. J. Chem. Phys. 1976, 64, 4006. 309 nm. (21) Tripathi, G. N. R.; Schuler, R. H. J . Chem. Phys. 1984, 81, 113. Previous tests of the FOCI calculations suggest that the (22) Stevens, W. J.; Basch, H.; Krauss, M. J . Chem. Phys. 1984,81,6026. theoretical predictions for the C6 and C5 H adduct spectra are (23) GAUSSIAN 9 0 Frisch, M. J.; Head-Gordon, M.; Trucks, G. W.; Foresman, J. B.; Schlegel, H. B.; Raghavachari, K.; Robb, M.; Binkley, J. S.; sufficiently accurate to call the experimental assignment for C5 Gonzalez, C.; Defrees, D. J.; Fox, D. J.; Whiteside, R. A.; Seeger, R.; Melius, adducts into question. The qualitative difference between the J. J.P.;Topiol,S.;Pople, C.F.;Baker, J.;Martin,R.L.;Kahn,L.R.:Stewart, C5 and C6 adduct spectra for both H and OH addition is quite J. A. Gaussian, Inc.: Pittsburgh, PA, 1990. certain. However, since these calculations are difficult and in an (24) Schmidt, M. W.; Baldridge, K. K.; Boatz, J. A.; Jensen, J. H.; Koseki, S.; Gordon, M. S.;Nguyen, K. A.; Windus, T. L.; Elbert, S . T. QCPEBull. early stage of development,we suggest that a careful examination 1990, 10, 52. of both the experimental analysis and calculation is in order for (25) Suppan, P. J. Photochem. Phorobiol., A: Chem. 1990, 50, 293. the visible transition predictions that are made. Since rather (26) Imabuko, K. J. Phys. Soe. Jpn. 1968, 24, 143. (27) Hayon, E. J. Chem. Phys. 1969, 51,4881. complicated kinetics occur in the radiolysis of uracil in solution, (28) Das, S.; Deeble, D. J.; Schuchmann, M.; von Sonntag, C. Znt. J. it is not surprising that another look at the assumptions and Radiat. Biol. 1984, 46, 7. deductions that both lead to and then utilize spectral assignments (29) Schuchmann, M. N.; Steenken, S.; Wroblewski, J.; von Sonntag, C. Int. J. Radiat. Biol. 1984, 46, 225. should be made. The present results are just a beginning toward
