Guanine Radical Reaction Processes: A Computational Description of

Nov 18, 2008 - Department of Physics and Astronomy, Georgia State University, P.O. Box 4106, Atlanta, Georgia ... Chem. B , 2008, 112 (51), pp 16908â€...
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J. Phys. Chem. B 2008, 112, 16908–16916

Guanine Radical Reaction Processes: A Computational Description of Proton Transfer in X-Irradiated 9-Ethylguanine Single Crystals Nayana Jayatilaka and William H. Nelson* Department of Physics and Astronomy, Georgia State UniVersity, P.O. Box 4106, Atlanta, Georgia 30302-4106 ReceiVed: July 15, 2008; ReVised Manuscript ReceiVed: September 20, 2008

Computational methods based on DFT procedures have been used to investigate proton-transfer processes in irradiated 9-ethylguanine crystals. Previous experimental results from X-irradiation and study of this system at 10 K found significant concentrations of two main products, R1, formed by N7-hydrogenation of the purine ring, and R2, the primary one-electron oxidation product (Jayatilaka, N.; Nelson, W. H. J. Phys. Chem. B 2007, 111, 7887). The objective of this work is to describe the processes leading to these products using computational methods that take into account molecular packing and bulk dielectric properties. The basic concept is that a proton will transfer following ionization if the net electronic energy of the system, consisting of the donor plus the acceptor plus any intervening molecules, becomes lower. Three approaches were used to investigate this concept, two based on energies computed for single molecules and one based on energies computed for two-molecule clusters arranged as in the crystals. The results are that the methods successfully predict the observed behavior, that it is energetically favorable on one-electron reduction for proton H1 to transfer from a neutral molecule to N7 of the neighbor, forming the N7-hydrogenated product, and that there is virtually no energy advantage for a proton to transfer upon one-electron oxidation. The results also support the proposal that the C8 H-addition radical, found only upon irradiation at 300 K, was the product of intramolecular transfer of the H7 proton to C8 in a process apparently requiring sufficient thermal energy for activation. Finally, the computations predict hyperfine couplings and tensors in very good agreement with those from experiment, thereby providing additional evidence for the success of the computations in describing the experimental observations. 1. Introduction Many important biochemical processes occur via proton transfers (PT) and are governed by the acidity (proton-donating character) and/or basicity (proton-accepting character) of the participants. A particular class of these is the set of processes initiated by one-electron oxidation or reduction of key molecular components within a system. One-electron oxidations and reductions are major events within a biological system upon exposure to ionizing radiation, and these events initiate a significant portion of the subsequent biochemical/biological effects of the exposure. One-electron ionization has a profound effect on the acidity/basicity of a molecule, and it thereby has a corresponding effect on its PT characteristics. For that reason, any comprehensive mechanistic description of radiation-initiated biological effects must include a description of the PT properties of key molecules following one-electron ionization. Guanine is a key component of DNA and has been the subject of many investigations over the past several decades. One reason is its now well-documented status as the DNA component with the lowest ionization potential. As such, guanine plays a central role in the initiation and progression of oxidative events affecting DNA. Of those seeking mechanistic descriptions of the chemistry underlying this, Steenken was among the first to offer a comprehensive view of PT between one-electron ionized DNA components.1,2 In doing so, he pointed out the importance of * To whom correspondence should be addressed. Phone: 404-413-5104. Fax: 404-413-5117. E-mail: [email protected].

these processes in determining the connection between the initial ionization event and the products important biochemically and biologically. Steenken’s approach consisted of measuring the pKa values of one-electron oxidized or reduced DNA components and using a comparison of these to discuss how the components might behave if ionized within the DNA strand. This approach recognizes that the probability of a transfer depends on the relative energy gain by the acceptor versus the loss by the donor (or vice versa); that is, the net result of the transfer should be a lower energy configuration. However, a limitation of the pKa method is that the measurements refer to the molecules as a whole and not to specific atomic sites within the molecules. This can be important for describing the behavior within DNA, where the components associate in specific ways. Thus, to remove this limitation characteristic of the standard pKa procedures, it is necessary to have well-defined systems exhibiting specific associations and to have procedures with which specific PTs can be detected. Single-crystal systems are well-defined and provide a variety of specific associations which may be used for experimental tests of PT models. In combination with single-crystal systems, EPR and ENDOR methods permit identifying specific proton interactions and thereby provide the potential for direct information on specific PTs. In addition, modern quantum chemistry methods, particularly those based on DFT procedures, provide a computational framework potentially suitable for a comprehensive description of PT processes. In fact, there have been a number of previous reports describing the probability of intermolecular PT between DNA components following ionization.3 However, none of these

10.1021/jp806262d CCC: $40.75  2008 American Chemical Society Published on Web 11/18/2008

Proton Transfer in X-Irradiated 9-Ethylguanine computational studies have been accompanied by experimental studies capable of providing direct information on the specifically proposed PTs. Recently, we completed an experimental study on crystals of the guanine derivative 9-ethylguanine4 (9-etG) in which the experimental results clearly identified the end products of PT arising from one-electron reduction. Slightly less clearly, the results also indicated the absence of PT following one-electron oxidation. Consequently, we decided to expand our work on this system to investigate the extent to which computational results can describe the experimentally observed PT behavior. The computational approach is to apply DFT methods to the description of possible intermolecular PT following one-electron ionization oxidation or reduction within the crystals. The crystallographic information provides a description of the intermolecular associations that are important components of the initial conditions. As well, because gas-phase computational results from isolated molecules omit the probably important role of the molecular surroundings, the point has been made repeatedly that isolated-molecule results are insufficient for describing processes in condensed media.5 Approaches to accounting for effects from the surrounding medium typically focus on water and employ some combination of treating it as a continuum dielectric or associating specific water molecules with the molecule of primary interest (microhydration). In this work, our objective is to describe processes in a homomolecular solid system (9-etG crystals) where the surrounding molecules, which are not water, contribute both a dielectric effect and an effect due to their specific associations with each other. Consequently, we will explore the various molecular forms in this work as isolated molecules, as molecules within a continuum dielectric medium, as molecules within a cluster of similar ones, and as clusters within a continuum dielectric medium. The primary focus is on the probability for various PTs following one-electron ionization, either reduction or oxidation, of a parent molecule within its environment. By comparing the computational results to those from experiment, it is possible to test the computational results for their capability to describe the experimental observations. 2. Methods Section Our basic approach was to calculate the energy difference between the initial ionized state and that after PT. Conceptually, this requires identifying a set of molecules that collectively undergo electron loss or gain and calculating the energy difference between their ionized states before and after PT. If the total energy of the final state is lower, then the transfer is thermodynamically probable, although the height of any barrier will control the kinetics. As well, the relative energy differences for the various possible transfers indicate the relative probabilities for occurrence. The major focus is on computational results from individual molecules. This parallels the discussion by Steenken in focusing on the differences between molecular pKa values for protonation and deprotonation. However, the major advantage of the computational procedure is that the energies for these processes can be calculated for specific molecular sites. Energy differences for possible PTs between two adjacent molecules in the crystal, following ionization of one to produce •M+/-, are given by the relations in eq 1 below. For reduction, the final products are a hydrogenated •(M + H) and a deprotonated (M - H)- molecule, while for oxidation, the products are a dehydrogenated •(M H) and a protonated (M + H)+ molecule. We note that these

J. Phys. Chem. B, Vol. 112, No. 51, 2008 16909 relations express Steenken’s acidity/basicity concept because they compare the protonation energy (basicity) of the acceptor to the deprotonation energy (acidity) of the donor.6

Reduction: ∆E ) {E[(M - H)-] + E[•(M + H)]} {E(M) + E(•M-)} Oxidation: ∆E ) {E[•(M - H)] + E[(M + H)+]} {E(M) + E(•M+)}

(1) Computational Methodology. All computations were performed with DFT methods as implemented in the Gaussian 03 (G03)7 suite of programs, and all made use of the B3LYP functional. The near absence of vertical overlap of 9-etG molecules in the crystals, as is shown below, virtually eliminates any concerns from the known weakness of DFT methods in describing stacking interactions. To explore effects from the dielectric properties of the surroundings, we optimized the geometries and computed the various energies in the vacuum state (ε )1.00) and by means of the IEFPCM method8 for chloroform (ε ) 4.90), acetone (ε ) 20.70), and water (ε ) 78.39). These computations provide one means of accounting for bulk electrostatic effects from the condensed environment; such effects are known to be important for charged species such as the •M+/- initial products. (We note that guanine films deposited on silicon exhibit optical birefringence with ε ∼ 3.5 at optical frequencies for directions lying in the molecular plane.9 At lower frequencies, the dielectric constant is likely to be higher due to guanine’s fairly large dipole moment.) The relations in eq 1 above for one-electron reduction explicitly include the energy of the 9-etG molecular anion. Computations on guanine anionic structures are well-known to be problematic because guanine’s electron affinity is small, such that electrons may associate with it only to form dipole-bound or otherwise highly diffuse states.10 For these cases, and particularly those for which the computations predict negative electron affinities, the reliability of the absolute energies is uncertain.11,12 In recent work, Puiatti et al.13 showed a highly linear relation between 1/ε and vertical electron affinities (VEA) computed for a series of molecules for which the VEA was known to be negative. This behavior, like that predicted by the Born equation,14 allowed them to use an extrapolation procedure to estimate the gas-phase VEA. Of relevance to this work is that the approach of Puiatti et al. provides a basis for assessing the reliability of the computed •M- energies relative to the parent M. Although it is a greater approximation to expect a linear relation between what amounts to adiabatic electron affinities and 1/ε, we found nearly exact linearity for the 9-etG anion computed in several variations, UB3LYP/6-311G(2df,3p)// 6-31G(d), UB3LYP/6-311+G(2df,3p)//6-31G(d), UB3LYP/6311G(2df,3p)//6-31+G(d), and UB3LYP/6-311+G(2df,3p)//631+G(d). (These results are shown in the Supporting Information.) For these, the major difference was between the single-point energy calculations at 6-311G(2df,3p) or 6-311+G(2df,3p). Because optimization at 6-31+G(d) versus 6-31G(d) had an insignificant impact on parent and anion energies, we concluded that satisfactory geometry optimizations did not require diffuse functions. Thus, we report results from optimizations and zeropoint energy calculations at 6-31G(d) with single-point energies computed at both 6-311G(2df,3p) and 6-311+G(2df,3p).15 All results include only electronic and zero-point energies and thereby express thermodynamic quantities at 0 K. However, we note that the related experiments were performed with the crystals irradiated at ∼10 K and studied without warming.4

16910 J. Phys. Chem. B, Vol. 112, No. 51, 2008 SCHEME 1

SCHEME 2

3. Experimental Background 3.1. Molecular Packing. As shown in Scheme 1, 9-ethylguanine molecules in the crystals form stacked two-moleculewide ribbons, interleaved such that alternate stacks lie parallel to the two equivalent but orthogonal 〈a〉 directions of the tetragonal crystal system.16 Within a ribbon, the virtually coplanar molecules are arranged in two-molecule units of “A” and “B” molecules. A and B molecules have slightly different geometries, and the hydrogen-bonding distances between those within a unit are slightly different (shorter) from those between one unit and another. The perpendicular stacks of ribbons are arranged such that the orthogonal 〈c〉 direction lies virtually in the plane of all guanine units. The ribbons are stacked such that the closest vertical spacing between any two is ∼3.5 Å with the B molecules in one layer over but offset ∼2.3 Å from the A molecules in the other. The net result is very little overlap of molecules in the stacked ribbons (Scheme 2). From the packing diagram, it is evident that the intermolecular proton transfers most likely to occur following one-electron ionization are those of H1 from one molecule to N7 of a neighbor, or from the amino of one molecule to O6 of a neighbor. 3.2. Magnetic Resonance Results. Exposure of the crystals to X-rays, a low LET radiation, leads to randomly distributed one-electron oxidized molecules, or “holes”, and free electrons. After loss of sufficient kinetic energy (by producing additional one-electron oxidized molecules and free electrons), the electrons become “trapped”. Trapping occurs either by the formation of molecular anions (one-electron reduced molecules) or by the electrons entering vacancies within the crystal made suitable for trapping by the electrostatic properties of the surrounding molecules.17 However, both electrons and holes are mobile within the system even after becoming associated with molecules. For example, there is extensive literature dealing with questions of the dynamics and the range of mobile charge centers (electrons and holes) within DNA.3 (The ease of electron and hole mobility within a system, and the relative energy associated with the respective trapping sites (“trap depth”), inevitably leads to (re)combination reactions and electron-hole neutralization. For this reason, the concentration of trapped, or “stabilized”, radicals usually is less, perhaps much less, than that of actual ionization events.) In a heteromolecular system

Jayatilaka and Nelson such as DNA, electrons are more likely to be trapped by molecules of higher electron affinity, and holes are more likely to be trapped by molecules of lower ionization potential. In a homomolecular system such as 9-etG, all molecules have the same electron affinity and ionization potential; thus, in such a system, the trapping of electrons and holes at specific molecules will be governed by the randomness of ionization events in combination with the barriers to recombination under the specific experimental conditions.18 During the period of transient processes following an ionization event, or at some time afterward, the surviving oxidation and reduction products, which are naturally paramagnetic, engage in typical chemical processes. The paramagnetic products detected by magnetic resonance are those remaining and stable under the experimental conditions after the transient processes have subsided. Important among the chemical processes are proton transfers, and they are the focus of the following discussion. Results from the 9-etG experimental work (at ∼10 K) indicated ionization-initiated formation of the N7-hydrogenated product and the primary oxidation product stabilized with no proton transfer. In the interest of the following discussion, it also is important to note that the results under these conditions showed no evidence for the products of net hydrogenation at O6 or dehydrogenation at the amino. Warming the crystals following irradiation led generally to disappearance of EPR spectra, indicating (re)combination reactions. Irradiation at room temperature, however, led to strong signals characteristic of the well-known C8 H-addition product of the guanine base along with signals from another product that could not be identified. With this background, the computational objective is to explore methods for describing PT mechanisms in 9-etG to account for the experimental observations in view of the molecular packing and the prospects for PT versus that of recombination. 4. Results and Discussion 4.1. 9-Ethylguanine Monomers. Table 1 shows the computed energies, relative to those of the parent in each of the chosen dielectric media, for the various molecular forms relevant to the following discussions. (Scheme 3 indicates the terminology “HO6a”, “HO6b”, etc. See the Supporting Information for a complete set of structures for the various tautomers.) We chose to include results from the 6-311G basis set, without diffuse functions, along with those from the 6-311+G set because of the problematic nature of guanine anions and the tendency of computations to lead to results “contaminated” with inappropriate diffuse character. Although it is unclear how to define the onset of this condition, it is usual to characterize it by visually examining the spatial extent of the valence electron density.12,13 Our approach has been to monitor the reported Mulliken spin distributions and corresponding hyperfine coupling values, and we have considered further examination necessary when a spin value greater than 1.0 is reported for any atom. For the anion calculations shown in the table, those using 6-311+G reported C2 spin values ranging from 1.24 in vacuum to 1.02 in water, while those from the 6-311G calculations ranged from 0.57 to 0.60 (vacuum to water). These differences indicate some degree of diffuse contamination in the 6-311+G results. It is interesting to note, however, that the relevant computed hyperfine couplings from the C2 spin (isotropic and dipolar couplings to a 13C nucleus at C2 and those to the amino protons) were within roughly 10% of the two different basis sets. Apparently, the increased spatial extent provided by the diffuse functions allows the valence electron density, on average, to be enough farther from the nuclei to approximately compensate for the additional unpaired spin density at C2.

Proton Transfer in X-Irradiated 9-Ethylguanine

J. Phys. Chem. B, Vol. 112, No. 51, 2008 16911

TABLE 1: Computed Electronic Energies, Relative to the Parent, for the Structures Indicated within the Various Dielectric Mediaa @ UB3LYP/6-311G(2df,3p)//6-31G(d)

@ UB3LYP/6-311+G(2df,3p)//6-31G(d)

(ε ) 1.00)

(ε ) 4.90)

(ε ) 20.70)

(ε ) 78.39)

(ε ) 1.00)

(ε ) 4.90)

(ε ) 20.70)

(ε ) 78.39)

vacuum

CHCl3

acetone

water

vacuum+

CHCl3+

acetone+

water+

0.0

0.0

0.0

+H7(+) +H3(+) +HO6a(+) +HO6b(+)

-996.1 -917.1 -931.6 -965.9

-1143.6 -1092.4 -1090.2 -1106.7

-1172.1 -1135.7 -1126.6 -1134.3

-H21(-) -H22(-) -H1(-)

1439.8 1458.8 1447.8

1308.3 1321.5 1298.4

1283.7 1293.5 1266.8

57.7

-72.5

-98.4

structure parent

anion +H3• +HO6a• +HO6b• +H7• +H8• cation -H21• -H22• -H1• -H91•b -H92•b -H101•b H1 to N3 H1 to O6a H1 to O6b H1 to N7 H21 to N3 H21 to O6a H21 to O6b H21 to N7 H22 to N3 H22 to O6a H22 to O6b H22 to N7 H-atom

-1357.3 -1369.7 -1369.7 -1401.5 -1443.2

-1358.7 -1364.8 -1363.9 -1395.6 -1444.2

-1360.1 -1364.6 -1362.9 -1394.2 -1444.7

703.9

557.8

528.6

0.0

0.0

0.0

0.0

0.0

Protonation -1178.4 -1148.6 -1136.3 -1141.9

-983.3 -904.8 -918.1 -951.8

-1129.8 -1080.4 -1074.7 -1090.2

-1157.7 -1124.0 -1110.4 -1116.5

-1164.0 -1137.4 -1120.0 -1124.0

Deprotonation 1278.0 1286.2 1258.8

1415.1 1433.9 1420.4

1285.7 1297.6 1272.0

1261.8 1269.6 1240.5

1256.5 1262.6 1232.9

28.7

-97.7

-123.3

-129.7

-1359.5 -1371.2 -1371.3 -1404.1 -1444.8

-1361.4 -1363.4 -1364.6 -1398.0 -1446.2

-1363.2 -1362.8 -1363.1 -1396.5 -1446.8

-1364.8 -1362.9 -1362.8 -1395.5 -1447.2

-105.8 Hydrogenation -1361.3 -1362.4 -1362.8 -1393.2 -1444.9 521.6

1675.7 1694.3 1695.4 1694.2 1687.7 1732.0

1688.9 1700.7 1700.0 1697.0 1688.6 1731.2

1694.7 1702.9 1702.1 1697.5 1688.8 1730.9

Dehydrogenation 1697.0 1703.7 1703.4 1697.3 1689.5 1731.8

81.1 2.7 4.3 74.0 61.4 111.7 87.4 95.5 53.9 138.3 111.7 114.8

59.8 18.0 23.4 52.5 55.0 105.6 94.4 78.2 51.5 122.7 109.6 91.2

48.3 23.9 30.6 46.1 51.4 102.1 96.8 73.6 49.7 113.2 107.3 82.9

Tautomers 43.4 26.0 31.7 44.5 49.4 101.1 96.5 72.4 48.4 109.8 104.8 80.1

-1318.4

-1318.5

-1318.6

-1318.6

716.7

571.8

542.9

535.8

1675.9 1694.1 1693.1 1694.1 1686.2 1730.0

1689.5 1700.5 1697.5 1697.2 1687.4 1729.3

1695.6 1702.6 1699.5 1697.7 1687.7 1729.0

1697.9 1703.3 1700.7 1697.4 1688.3 1729.7

78.3 4.6 5.9 69.7 60.8 112.7 88.8 93.5 53.3 139.3 112.9 112.4

54.8 21.9 26.9 47.3 53.6 106.9 96.7 75.6 50.0 123.1 111.0 87.5

42.1 28.7 35.1 40.6 49.4 103.7 99.9 70.9 47.8 113.1 108.7 78.6

36.5 31.0 36.3 39.0 46.9 102.6 99.6 69.8 46.2 109.5 106.2 75.8

-1318.4

-1318.5

-1318.6

-1318.6

a The correspondingly computed H-atom values are shown for reference, and all energies are kJ/mol. hydrogens of the ethyl, and H101 is one from its methyl group.

SCHEME 3

Some general observations from the data in Table 1 are (1) placement of the formally charged structures within a dielectric medium lowers their energy (relative to the parent) by an amount increasing with dielectric constant, with the major effect occurring at relatively low ε (i.e., qualitatively as predicted by the Born equation), (2) the neutral species, from hydrogenation or dehydrogenation, are relatively unaffected, with a slight lowering for hydrogenation and a slight raising for dehydrogenation with increasing ε, (3) the tautomerically rearranged structures exhibit energies generally decreasing with increased dielectric constant, but with a few exceptions involving protonation of O6,19 and (4) in addition, relative to those of the

b

H91 and H92 are the methylene

respective neutral parents, energies computed at 6-311+G (with diffuse functions) behaved with a net dependence on formal charge in relation to those to those computed at 6-311G (without diffuse functions) as follows. For negatively charged species, they were lower (by ∼25 kJ/mol), for positively charged species, they were higher (by ∼15 kJ/mol), and for neutral species, they were changed very little (by ∼(2 kJ/mol). Overall, the energies varied smoothly and monotonically with increasing dielectric constant. Finally, we note that the vacuum-state deprotonation energies (at 6-311+G) are nearly the same as those reported previously for 9-H guanine20 and that the protonation energies for the 9-ethyl derivative are greater in magnitude by ∼15-20 kJ/mol. Notable from the results is the prediction that the electron affinity of the anion becomes positive (EA ) -∆E, shown in Table 1) within a medium of ε < 4.90 (i.e., at ε ∼1.5 as indicated by the plots in the Supporting Information). The results also indicate the high “driving force” for anion-cation recombination, ∼750 kJ/mol in vacuum, decreasing to ∼400 kJ/mol in water. In order for radiation-initiated products to be stabilized long enough for typical chemistry (and to allow magnetic

16912 J. Phys. Chem. B, Vol. 112, No. 51, 2008

Jayatilaka and Nelson

TABLE 2: Net Proton-Transfer Energies for the Two- and Three-Molecule Processes Initiated by One-Electron Reduction or Oxidation (All Energies Are in kJ/mol) @ UB3LYP/6-311G(2df,3p)//6-31G(d)

@ UB3LYP/6-311+G(2df,3p)//6-31G(d)

Two-Molecule: (•M-) + M f •(M + H) + (M - H)Reduction:

Vacuum

CHCl3

Acetone

Water

Vacuum

CHCl3

Acetone

Water

result

(ε ) 1.00)

(ε ) 4.90)

(ε ) 20.70)

(ε ) 78.39)

(ε ) 1.00)

(ε ) 4.90)

(ε ) 20.70)

(ε ) 78.39)

-11.3 12.3 -53.0

-24.7 17.0 -73.4

-29.0 19.2 -79.4

-28.6 21.0 -80.3

-12.5 15.1 -53.2

-28.3 18.8 -76.5

-32.7 22.0 -83.1

-32.9 23.5 -84.7

+H7• +HO6b• +H8•

Three-Molecule:a (•M-) + M + M f •(M + H) + Mt + (M - H)Reduction:

Vacuum

CHCl3

Acetone

Water

Vacuum

CHCl3

Acetone

Water

result

(ε ) 1.00)

(ε ) 4.90)

(ε ) 20.70)

(ε ) 78.39)

(ε ) 1.00)

(ε ) 4.90)

(ε ) 20.70)

(ε ) 78.39)

-15.1 62.6 115.8 99.6

8.6 27.8 85.2 111.3

18.5 17.1 75.9 116.0

22.4 15.9 74.2 117.5

-11.8 57.2 113.7 103.9

12.3 19.0 80.7 115.6

23.6 7.9 71.6 121.9

27.1 6.1 69.6 123.0

+H7• (1) +H7• (2) +HO6b• (1) +HO6b• (2)

Two-Molecule: (•M+) + M f •(M - H) + (M + H)+ Oxidation:

Vacuum

CHCl3

Acetone

Water

Vacuum

CHCl3

Acetone

Water

result

(ε ) 1.00)

(ε ) 4.90)

(ε ) 20.70)

(ε ) 78.39)

(ε ) 1.00)

(ε ) 4.90)

(ε ) 20.70)

(ε ) 78.39)

-3.7 6.8 990.4

-1.4 24.4 1142.9

1.4 31.8 1174.3

3.5 33.5 1182.1

-6.0 8.2 977.4

-4.1 11.1 1128.7

-1.1 36.1 1159.6

0.9 38.0 1167.5

-H1• -H21• -H22•b

Three-Molecule:a (•M+) + M + M f •(M - H) + Mt + (M + H)+ Oxidation:

Vacuum

CHCl3

Acetone

Water

Vacuum

CHCl3

Acetone

Water

result

(ε ) 1.00)

(ε ) 4.90)

(ε ) 20.70)

(ε ) 78.39)

(ε ) 1.00)

(ε ) 4.90)

(ε ) 20.70)

(ε ) 78.39)

70.2 121.9 94.1 -19.0

51.1 113.7 118.7 10.9

47.5 112.8 128.6 24.6

48.0 112.4 130.0 28.7

63.6 118.8 96.9 -17.4

43.2 111.1 124.3 14.9

39.5 110.9 136.0 30.1

39.9 110.6 137.6 34.4

-H1• (1) -H1• (2) -H21• (1) -H21• (2)

a “Mt” indicates the tautomer formed by the three-molecule transfer. b The process leading to -H22• is proton loss from the one-electron oxidation product rather than proton transfer to a neighboring molecule. This entry is included to emphasize that it is likely to occur only with a source of additional energy or an energy-compensating process elsewhere.

resonance observation), it is necessary for the system to present energy barriers sufficient to overcome this large potential energy for recombination. The discussions below focus mainly on the behaviors of the primary one-electron reduced or oxidized products. It is useful to note that the results predict that the one-electron oxidized product is unlikely to undergo spontaneous deprotonation. Specifically, the deprotonation energy of the cation, E[(•M H)] - E[•M+], is positive for all three NH sites, ∼990 kJ/mol in vacuum increasing to ∼1175 kJ/mol in water. In fact, the deprotonation energy of the cation and that of the neutral parent approach each other as ε increases. For example, deprotonation of the parent at N1 requires 456 kJ/mol more energy than the cation in vacuum but only 77 kJ/mol more in water. The possibility of diffuse, or dipole-bound, character of the guanine reduction product makes discussions of it somewhat problematic. With that caveat, the results shown in Table 1 predict that the one-electron reduction product exhibits a very strong tendency to protonate spontaneously. For example, the protonation energy, E[(•M + H)] - E[•M-], of the anion at N7 ranges from ∼1450 kJ/mol in vacuum to ∼1275 kJ/mol in water. 4.2. Proton-Transfer Energies. The basic concept of this investigation is that the initial one-electron ionized products engage in PT with surrounding molecules and that PT occurs on a competitive basis; that is, it will occur if there is an energy “benefit” to the collective set of molecules involved, and the

most likely result is that giving the greatest benefit. Upon occurrence, deprotonation of the oxidation product leads to paramagnetic dehydrogenated radicals and neighboring diamagnetic positive ions (resulting from protonation), while protonation of the reduction product leads to paramagnetic hydrogenated radicals and neighboring diamagnetic negative ions (resulting from deprotonation). Table 2 shows energy differences calculated from the data in Table 1 according to eq 1 above. For reduction, transfer of the H1 proton from a neutral neighbor to N7 of the anion forms the N7-hydrogenated product and lowers the energy for all four dielectric values. Transfer of the amino proton H21 from a neutral neighbor to O6 of the anion forms the O6-hydrogenated product but raises the energy for all dielectric media. Thus, the results predict that N7-protonation of the anion (i.e., net hydrogenation at N7) is much more highly favored than that of O6 hydrogenation. In addition, the transfer of H1 to N7 following reduction is predicted to be increasingly favorable as the dielectric constant increases. This prediction is consistent with the experimental results from 9-etG. (The table also shows the much deeper trap provided by the C8hydrogenated structure originating from reduction, a point that will be discussed further below.) For oxidation, transfer of the H1 proton to N7 of the neutral neighbor, forming the N1-dehydrogenated product, slightly lowers the energy in vacuum (-3.7 kJ/mol) and chloroform (-1.4 kJ/mol) and slightly raises it in acetone (+1.4 kJ/mol) and water (+3.5 kJ/mol). Following oxidation, transfer of the

Proton Transfer in X-Irradiated 9-Ethylguanine

J. Phys. Chem. B, Vol. 112, No. 51, 2008 16913

SCHEME 4: Reduction

amino proton H21 to O6 of a neutral neighbor (i.e., dehydrogenation of the amino group) raises the energy for all dielectric values. Thus, the results predict that deprotonation of the cation at N1 via transfer of H1 to a neighboring N7 has, at best, a marginal likelihood of occurring or remaining if it occurs. As well, they predict that deprotonation of the cation via transfer of amino proton H21 to O6 of a neighbor is unlikely. Again, these predictions are consistent with the experimental results. (The table also lists the large energy increase associated with dehydrogenation at H22; thus, it appears highly unlikely that the process of oxidation f deprotonation by H22 will be a spontaneous event. We note, however, that this does not include the “solvation” energy of the “free” proton due to its interaction with the dielectric reaction field.) 4.3. Proton Transfers Involving Three Molecules. Table 2 also indicates results for a three-molecule transfer. Previous discussions considered a sequence of proton transfers based on the concept that a separation of spin and charge, as far as possible, would lead to the lowest energy of the system.21 To explore the possibility that such a process could enhance the probability of forming hydrogenated or dehydrogenated products, we investigated possible routes for forming the N7- and O6-hydrogenated products following reduction and those of forming the N1- and amino-dehydrogenated products following oxidation. For this purpose, it was sufficient to consider arrangements involving only three molecules. In these cases, the molecule at one end of each group is the paramagnetic species, that at the other end carries the charge, and the middle molecule takes on an appropriate tautomeric form of the parent.

In all cases, the rearrangements involve only those of hydrogens participating in hydrogen bonds between the molecules. As is shown in Schemes 4 (reduction) and 5 (oxidation), there are two possible final arrangements for each case, reduction with hydrogenation at N7, +H7• (1), and +H7• (2); reduction with hydrogenation at O6, +H6b• (1), and +H6b• (2); oxidation with dehydrogenation at N1, -H1• (1), and -H1• (2); and oxidation with dehydrogenation at the amino, -H21• (1), and -H21• (2). The results in Table 2 indicate that only two of these, +H7• (1) and -H21• (2), lead to energies lower than that of the initial arrangement following reduction/ oxidation. In addition, for these two arrangements, the “transferred” configuration is more stable only for the vacuum state. To separate the spin and charge farther requires forming additional tautomers from intervening molecules; however, all tautomers listed in Table 1 are higher in energy than the parent. This leads to the prediction that the change in energy of the system will become increasingly positive, and the arrangement increasingly unlikely, as the number of intervening tautomers increases. On that basis, this analysis predicts that the separation of spin and charge in the 9-etG system will be at most that of their being located on adjacent molecules. 4.4. Intramolecular Proton Transfer from N7 to C8. The results in Table 2 indicate the much more stable configuration resulting from hydrogenation at C8 (∼40 kJ/mol lower than the N7-hydrogenated form and ∼1450 kJ/mol lower than the parent in vacuum). The C8 hydrogen-addition products of purines are well-known22 and are generally thought to occur both by hydrogen atom addition and by protonation of the

16914 J. Phys. Chem. B, Vol. 112, No. 51, 2008

Jayatilaka and Nelson

SCHEME 5: Oxidation

primary reduction product.1 In the 9-etG experimental work, we found no spectroscopic evidence for the presence of the C8 H-adduct following irradiation at 10 K, nor was there any evidence for its formation upon controlled warming of the crystals irradiated at low temperatures. However, spectral features of this product were prominent following irradiation at room temperature. Thus, we sought a transition structure that might connect the N7-hydrogenation product observed at 10 K to the C8 H-adduct observed at room temperature. The QST2 method available in G03 led to such a structure, and it was confirmed by subsequent IRC calculations as a connection between the N7- and C8-hydrogenated structures with forward and reverse barrier heights of 178.8 and 218.7 kJ/mol, respectively. (More details on these results are shown in the Supporting Information; also, we note that Yang and Qi23 considered the similar process for guanine and related purines but involving transfer of arylnitrenium ions instead of a proton.) The high barrier separating the N7- and C8-hydrogenated structures is consistent with the observation that the C8 H-adduct did not form upon controlled warming; the (re)combination reactions could be initiated with lower activation energy. As well, formation of the C8 H-adduct upon irradiation at room temperature is consistent with the notion that thermal energy (at 300 vs 10 K) during the irradiation allowed a population of the adducts to form. The barrier height (∆E/k ∼ 2 × 104 K) predicts that the transfer rate will be low if the process is intermolecular. However, because it is an intramolecular transfer, thermal excitation of appropriate vibrational modes within the N7-hydrogenated molecule may enable the transfer at 300 K.

(For example, with the N7-hydrogenated pretransfer structure, G03 predicts the third most intense vibrational mode, occurring at ∼500 cm-1, to be one that involves rocking of both the N7-H7 and C8-H8 bonds. The most intense mode occurs at a much higher frequency, ∼1850 cm-1.) 4.4. Two-Molecule Clusters. The discussion above is based only on properties of single molecules and omits the potentially important element of the interactions between molecules. These interactions of course will change upon ion formation, and the change might be decisive in determining the subsequent processes. To explore the importance of this effect at a basic level, we performed a series of computations employing twomolecule clusters with initial geometries established by A and B molecule crystal coordinates. We acknowledge the possible introduction of “end effects” by limiting the cluster only to two molecules, a choice made for reducing the computational load. We note that preliminary computations using four-molecule clusters, in which the coordinates of the outer two molecules were frozen, also predicted transfer of H1 to N7 upon oneelectron reduction to be a stable configuration. Without further investigation, we considered it problematic to make quantitative use of energies from such computations, however. Table 3 shows the energies (relative to those of the respective neutral parents) computed for oxidation and reduction products, with and without proton transfers, in the various dielectric media. For both reduction and oxidation in the vacuum state, the transfer of amino proton H21 to O6 was an unstable configuration in that the proton returned to the amino nitrogen during optimization. In addition, for oxidation, the H21-to-O6 transfer was

Proton Transfer in X-Irradiated 9-Ethylguanine

J. Phys. Chem. B, Vol. 112, No. 51, 2008 16915

TABLE 3: Computed Energies, Relative to the Parent, for the A/B Molecule Clusters in 9-etG (All Energies Are in kJ/mol)a @ UB3LYP/6-311G(2df,3p)//6-31G(d)

@ UB3LYP/6-311+G(2df,3p)//6-31G(d)

(ε ) 1.00)

(ε ) 4.90)

(ε ) 20.70)

(ε ) 78.39)

(ε ) 1.00)

(ε ) 4.90)

(ε ) 20.70)

(ε ) 78.39)

structure

vacuum

CHCl3

acetone

water

vacuum

CHCl3

acetone

water

AB parent AB anion AB anion, H1 f H7 AB anion, H21 f HO6b AB cation AB cation, H1 f H7 AB cation, H21 f HO6b Etransfer - Eion: AB anion, H1 f H7 AB cation, H1 f H7

0.0 -6.0 34.0 returned 601.5 614.2 returned

0.0 -96.7 -91.1 -63.7 521.2 529.4

0.0 -109.3 -125.8 -86.9 508.0 515.6

0.0 -117.0 -136.0 -99.7 506.5 513.2

0.0 -27.4 10.9 returned 615.4 627.4 returned

0.0 -112.6 -117.1 -85.4 535.9 542.8

0.0 -132.9 -149.6 -108.8 522.9 529.1

0.0 -140.5 -160.1 -121.0 521.3 526.3

5.6 8.2

-16.5 7.6

-19.0 6.7

-4.5 6.9

-16.7 6.2

-19.6 5.0

40.0 12.7

38.3 12.0

a The counterpoise procedure is not implemented in G03 for PCM calculations. However, for the vacuum case, the BSSE differences (from the parent) at 6-311G(2df,3p) for the anion and the H7-transferred anion were +5.5 and +9.7 kJ/mol, respectively; the respective differences with 6-311+G(2df,3p) were negligible. The values shown in the table do not include a BSSE correction.

similarly unstable in all media. Otherwise, the computed energies for the clusters are qualitatively related to the dielectric constant and the use of diffuse functions in roughly the same way as those of the single molecules in Table 1. Energies in all cases for the basic reduction product are predicted to be lower than those of the parent, indicating the two-molecule unit to be a stable trapping site for an electron. Thus, the AB cluster has a positive electron affinity with a value greater for each ε than that calculated for the individual molecules. The bottom two lines of Table 3 compare the preand post-transfer energies for the one-electron ionized clusters. In vacuum and chloroform, the results indicate that subsequent transfer of H1 to N7 raises the energy of the one-electron reduced cluster (∆E ) +40.0 and +5.6 kJ/mol, respectively), while the energy is lowered when the transfer occurs in acetone and water (∆E ) -16.5 and -19.0 kJ/mol, respectively); thus, the computations predict that conditions are favorable for PT, following reduction, in the media with higher ε. Also, for all media, the results predict that transfer of H1 to N7 raises the energy of the one-electron oxidized cluster and thus that the conditions are unfavorable for PT following oxidation. Unfortunately, we were unable to locate transition structures connecting the pre- and post-transfer structures for either the reduction or oxidation cases; thus it was not possible to identify or quantify the barriers to the transfers.24 (Nonetheless, it is evident that barriers exist on the potential energy surface; otherwise the pre- and post-transfer structures would not have been stable during geometry optimization.) 4.5. Computed versus Experimental Hyperfine Couplings. A key test for the computational procedures is their success in predicting the hyperfine coupling parameters, magnitudes, and dipolar directions observed in the EPR/ENDOR experiments. It is important to note that the single-crystal procedures provide coupling tensors and thereby provide results capable of testing the predicted molecular geometries of the radicals. It is difficult to envision that computed energy values can be correct unless the corresponding geometries also are correct. Hyperfine couplings of the oxidation product (radical R2) are represented well by the single-molecule and AB cluster calculations (see Tables S4 and S5 of the Supporting Information). Single-molecule computations on the N7-hydrogenated structure reasonably approximate the observed hyperfine couplings but reflect an overestimated degree of bending by the C8-H8 and N7-H7 bonds. (This is reflected in the magnitude and sign of the respective R-couplings’ isotropic components;25 see Tables S2 and S3 of the Supporting Information.) The overestimate

for the C8-H8 bending is essentially corrected by the calculations in acetone and water, but the poor description of the N7-H7 bond geometry remains. For the two-molecule AB cluster (Table S3 of the Supporting Information), the computation for the vacuum state completely fails to describe the hyperfine couplings. In contrast, the AB computations in all dielectric media do an outstanding job of describing the geometries of the three couplings in the radical. The magnitudes of the computed isotropic components differ from the experimental values by ∼10%; however, these values are extremely difficult to reproduce accurately and depend on the combined choice of functional and basis set. (Because they are less sensitive to this computational choice, the magnitudes and directions of the dipolar hyperfine components provide a more stable test of the computational procedures.) Thus, insofar as the hyperfine couplings are concerned, the quality of the computational description is significantly improved by taking into account the molecular and dielectric surroundings of the radicals, an approach actively pursued by Pauwels, Waroquier, and their colleagues.26 5. Summary and Conclusions In summary, therefore, the computational results from the individual-molecule models and the two-molecule AB clusters are consistent with, and describe well by, the experimental observations as follows: 1. One-electron reduction leads to PT from N1 of one molecule to N7 of another, thereby forming the N7-hydrogenated structure detected by EPR/ENDOR in 9-etG; this was detected by EPR/ENDOR and predicted computationally by the energy results. 2. One-electron oxidation does not lead to PT in 9-etG (meaning that the EPR/ENDOR results detected the primary charged product); this was detected by EPR/ENDOR and predicted computationally by the energy results. 3. Irradiation at 10 K does not produce a detectable concentration of the C8 H-addition radicals, nor does controlled warming lead to their appearance; this was detected by EPR/ ENDOR and is consistent with computed energy results. 4. Irradiation at room temperature (∼300 K) leads to a significant concentration of the C8 H-adducts, suggesting their formation from the N7-hydrogenated product via an intramolecular PT that requires sufficient thermal “activation” as a prerequisite; this was detected by EPR/ENDOR and is consistent with computed energy results.

16916 J. Phys. Chem. B, Vol. 112, No. 51, 2008 The computational results also emphasize the importance of taking into account the molecular surroundings, that is, the dielectric properties and molecular associations, when using computational models for interpreting and/or predicting experimental results. Detailed EPR/ENDOR results from single crystals provide a stringent test for the capabilities of current computational methods to describe the hyperfine couplings and thus to describe the molecular geometry. Therefore, the consistency between the computational and experimental results in 9-etG (i.e., the products predicted by the energy calculations along with their magnetic hyperfine coupling magnitudes and dipolar tensors) provides an important validation of their mutual support for each other in general. From this, we may conclude that these experimental and computational procedures, if applied in combination, have the capability of providing a detailed and computation-based description of the PT properties of molecules following one-electron ionization. Acknowledgment. Portions of this work were made possible with computer time provided by the Georgia State University High Performance Computing Core Facilities. Supporting Information Available: Figures S1 and S2 showing the energies versus ε and 1/ε for the one-electron reduced structures, Figure S3 showing the results of the IRC calculation for the transfer of H7 to H8, Table S1 showing the structures of all of the tautomers listed in Table 1, and Tables S2-S5 showing the experimental versus computed hyperfine couplings for the radicals observed at 10 K. This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Steenken, S. Chem. ReV. 1989, 89, 503. (2) Steenken, S. Free Radical Res. Commun. 1992, 16, 349. (3) Cai, Z.; Sevilla, M. D. Top. Curr. Chem. 2004, 237, 103. (4) Jayatilaka, N.; Nelson, W. H. J. Phys. Chem. B 2007, 111, 7887. (5) (a) Hunter, K. C.; Wetmore, S. D. J. Phys. Chem. A 2007, 111, 1933. (b) Naumov, S.; von Sonntag, C. Radiat. Res. 2008, 169, 364. (6) The notation •(M-H) indicates hydrogen loss; that is, •(M-H) ) (M)-(•H). Similarly, (M-H)- indicates deprotonation or (M-H)- ) (M)(H+). Likewise, •(M + H) and (M + H)+, respectively, indicate hydrogenation and protonation. (7) Frisch, M. J. Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr.; Vreven, T.; Kudin, K. N.; Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.; Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.; Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda, Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.; Bakken, V.; Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.; Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.; Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.; Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.; Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.; Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz,

Jayatilaka and Nelson P.; Komaromi, I.; Martin, R. L.; Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe, M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A. Gaussian 03, revision D.04; Gaussian, Inc.: Pittsburgh, PA, 2003. (8) Cances, E.; Mennucci, B.; Tomasi, J. J. Chem. Phys. 1997, 107, 3032. (9) Hinrichs, K.; Silaghi, S. D.; Cobet, C.; Esser, N.; Zahn, D. R. T. Phys. Status Solidi B 2005, 242, 2681. (10) Roehrig, G. H.; Oyler, N. A.; Adamowicz, L. Chem. Phys. Lett. 1994, 225, 265. (11) Vera, D. M. A.; Pierini, A. B. Phys. Chem. Chem. Phys. 2004, 6, 2899. (12) Li, X.; Cai, Z.; Sevilla, M. D. J. Phys. Chem. A 2002, 106, 1596. (13) Puiatti, M.; Vera, D. M. A.; Pierini, A. B. Phys. Chem. Chem. Phys. 2008, 10, 1394. (14) Born, M. Z. Phys. 1920, 1, 45. (15) To obtain fully completed geometry optimizations in some cases, it was necessary to use the “OPT)CALCALL” option; accordingly, all geometries and frequencies for the individual-molecule cases were obtained with this approach. (16) Destro, R.; Kistenmacher, T. J.; Marsh, R. E. Acta Crystallogr., Sect. B 1974, 30, 79. (17) Box, H. C.; Budzinski, E. E.; Freund, H. G. J. Chem. Phys. 1978, 69, 1309. (18) Bernhard, W. A.; Mroczka, N.; Barnes, J. Int. J. Radiat. Biol. 1994, 66, 491. (19) We note that all absolute energies decreased with increasing ε, as expected. The increased relative energies shown in Table 1 for some molecules reflect the magnitude of their dipole moments in comparison to that of the parent. When placed within a dielectric, the energy of a neutral molecule will decrease in proportion to the square of its dipole moment (Onsager’s model of the dipole reaction field). Thus, with increasing ε, the parent’s energy will decrease more rapidly than that of another neutral form with a smaller dipole moment; see: Onsager, L. J. Am. Chem. Soc. 1936, 58, 1486. (20) (a) Chandra, A. K.; Nguyen, M. T.; Uchimaru, T.; ZeegersHuyskens, T. J. Phys. Chem. A 1999, 103, 8853. (b) Huang, Y.; Kenttamaa, H. J. Phys. Chem. A 2004, 108, 4485. (c) McConnell, T. L.; Wheaton, C. A.; Hunter, K. C.; Wetmore, S. D. J. Phys. Chem. A 2005, 109, 6351. (21) (a) Bernhard, W. A.; Barnes, J.; Mercer, K. R.; Mroczka, N. Radiat. Res. 1994, 140, 199. (b) Nelson, W. H.; Sagstuen, E.; Hole, E. O.; Close, D. M. Radiat. Res. 1998, 149, 75. (22) (a) Shields, H.; Gordy, W. Proc. Natl. Acad. Sci. U.S.A. 1959, 45, 269. (b) Herak, J. N.; Gordy, W. Proc. Natl. Acad. Sci. U.S.A. 1965, 54, 1287. (c) Alexander, C., Jr.; Gordy, W. Proc. Natl. Acad. Sci. U.S.A. 1967, 58, 1279. (23) Yang, Z.-Z.; Qi, S.-F. J. Phys. Chem. B 2007, 111, 13444. (24) We found a candidate for the transition structure connecting the pre- and post-transferred geometries of the reduction product in water that suggested forward and reverse barriers of 10.0 and 30.6 kJ/mol, respectively. These indicate a forward barrier that is quite low (∼2.4 kcal/mol). Unfortunately, we were unable to confirm this as the correct transition structure because the IRC calculation did not go to completion. (25) Erling, P. A.; Nelson, W. H. J. Phys. Chem. A 2004, 108, 7591. (26) (a) Pauwels, E.; Van Speybroeck, V.; Lahorte, P.; Waroquier, M. J. Phys. Chem. A 2001, 105, 8794. (b) Pauwels, E.; Van Speybroeck, V.; Waroquier, M. Int. J. Quantum Chem. 2003, 91, 511. (c) Pauwels, E.; Van Speybroeck, V.; Vanhaelewyn, G.; Callens, F.; Waroquier, M. Int. J. Quantum Chem. 2004, 99, 102. (d) Declerck, R.; Pauwels, E.; Van Speybroeck, V.; Waroquier, M. Phys. ReV. B 2006, 74, 245103/1. (e) Pauwels, E.; Van Speybroeck, V.; Waroquier, M. J. Phys. Chem. A 2004, 108, 11321.

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