**2. Nucleobase oxidation via ionizing radiation**

Ionizing radiation can potentially be absorbed by any of the three nucleotide components of DNA (i.e., phosphate, sugar or nucleobase) or its surrounding waters; also an essential part of DNA structure.(Kumar & Sevilla 2010) Primary damage of, for example, nucleobases, is caused by their direct absorption of radiation. Secondary damage (such as that described later in this chapter) can also occur, for instance, when the radiation is absorbed by the solvent, thus generating radicals or solvated electrons which then attack the nucleobase.(von Sonntag 1987; von Sonntag 1996)

Direct absorption (primary damage) can cause the formation of a radical-cationic base via the loss of an electron, i.e., generation of an electron hole. Due to the stacking of nucleobases within DNA charge transfer (transfer of the hole) can then occur along the strand. Consequently, this enables 'primary damage related' reactions to occur distant from the site of initial damage. The potential for charge transfer due to the *π*–orbital interactions between bases was proposed as early as 1962. (Eley & Spivey 1962) Alternatively, however, it may enable charge recombination to occur further along the chain. This is because radical-anionic bases can be formed by the capture of the free electrons, where the resulting damage to the nucleobase also constitutes primary damage. Guanine has the lowest ionization potential of

Mechanisms of Mutagenic DNA Nucleobase Damages and

(aq) –e–

ionized, in agreement with experimental observations.

–e–

(i.e., C/T(aq) + e–

mol–1, respectively).

(vac) –e–

Their Chemical and Enzymatic Repairs Investigated by Quantum Chemical Methods 393

B•–(aq) B(aq) B•+(aq)

A•– 40.7 2.3 –59.3 (–56.0)a A 136.7 98.3 36.7 (32.7)c A•+ G•– 37.9 –0.5 –62.1 (–63.2)a G 125.9 87.5 25.9 (29.7)c G•+ T•– 51.9 13.5 –48.1 (–25.6)b T 145.2 106.8 45.2 (39.2)c T•+ C•– 43.3 9.9 –51.7 (–25.8)b C 147.6 109.2 47.6 (36.9)c C•+ Table 2. Calculated (see text) standard free energies (in kcal mol–1) of primary ionizations of the four nucleobases in aqueous solution. Experimental values are in parenthesis and taken from references: a (Seidel et al. 1996), b (Steenken et al. 1992), c (Steenken & Jovanovic 1997). ET is a common process that occurs upon absorption of radiation by nucleobases. Using a first principles approach the free energy changes involved with such a process for all four DNA nucleobases were calculated and are shown in **Table 2**.(Llano & Eriksson 2002) It can be seen that the ionization of each of the anionic bases (B•–(aq)) can be either endothermic or exothermic depending on the reference state of the electron. For example, in the vacuum or aqueous state the oxidation of the anionic bases is generally an endothermic process. The only exception occurs for guanine in the aqueous state in which the process is marginally exothermic (–0.5 kcal mol–1). In contrast, in the case of the SHE reference state, oxidation of each base is markedly exothermic. For A•– and G•– the values calculated are in close agreement to those obtained experimentally.(Seidel et al. 1996; Steenken & Jovanovic 1997; Steenken et al. 1992) In contrast, those calculated for C•– and T•– are not in as good agreement, being almost twice the corresponding experimental values. However, the overall trends are consistent; the oxidation of C•– or T•– is thermodynamically less favorable than that of A•– or G•–. Conversely, the reverse process, capture of a solvated electron by C or T

(vac) –e–

(aq) C/T•–(aq)) is thermodynamically preferred (–9.9 kcal mol–1 and –13.5

kcal mol–1, respectively) compared to that involving A or G (–2.3 kcal mol–1 and 0.5 kcal

Oxidation of the neutral bases is calculated to be endothermic for each reference state of the electron (**Table 2**). The degree of endothermicity, however, depends on the reference state being most endothermic in vacuum and least for the SHE reference state. Unlike that observed for the radical anion bases, the SHE calculated values for the neutral bases are all in good agreement with experiment. The largest difference occurs for C and is now only 10.7 kcal mol–1 compared to the 25.9 kcal mol–1 difference for C•–, while the smallest difference (– 3.8 kcal mol–1) is observed for guanine. In addition, neutral G is calculated to have the lowest free energy of oxidation and is thus the easiest of the four DNA nucleobases to be

The free energies associated with the loss of a proton from the resulting radical-cationic bases to solution were calculated and are given in **Table 3**. For A/G•+ the energy changes associated with deprotonations to form *syn*–A(N6–H), *anti*–A(N6–H), and G(N1–H) were determined to be quite small at just –0.6, –0.7 and –0.3 kcal mol–1 respectively.(Llano & Eriksson 2004a) In contrast, the energy changes associated with deprotonation of C/T•+ to give *syn–*C(N4–H) or *anti–*C(N4–H) and T(C5–H) are larger at –5.2, –4.5 and –22.3 kcal mol–1, respectively.(Llano & Eriksson 2004a) It is noted that T(C5–H) has only been observed in the solid state and not in solution,(Steenken 1989) and thus will not be discussed herein. Unlike the other deprotonation processes, formation of T(N3–H) was found to be endothermic and

(aq) –e–

(SHE)

(SHE) –e–

the four DNA nucleobases, followed by adenine.(Hush & Cheung 1975; Kumar & Sevilla 2010; Steenken & Jovanovic 1997; Yang et al. 2004) Consequently, they are in general oxidized to give their radical-cations while thymine and cytosine act as electron sinks and form their radical-anions. However, vice versa, in the transfer of electron holes guanine typically acts as a sink for DNA radiative oxidation.(Cadet et al. 2008; Kumar & Sevilla 2010) Reduction/oxidation of a nucleobase can significantly affect its properties. In particular, it has been shown that their oxidation greatly increases their acidity (lowers their pKa).(Kumar & Sevilla 2010) Indeed, the formation of a radical base is often associated with proton transfer reactions that can lead to further nucleobase damage. However, as has also been noted, these same proton transfers can result in nucleobase repair.(Llano & Eriksson 2004b)

Fig. 1. Structures of the dehydrogenated nucleobases for both the cations and anions. The position of the missing hydrogen is marked by the asterisk.

Unfortunately, our understanding of these important electron- (ET) and proton-transfer (PT) reactions is incomplete.(Llano & Eriksson 2004a) This is not only due to the highly transient and reactive nature of key species involved, but also because experimental measurements of free energies for such processes can only be obtained indirectly.(Llano & Eriksson 2004a) Moreover, at times, the available experimental techniques (photoelectron spectroscopy, mass spectroscopy, pulse radiolysis and electrochemical techniques) appear to give contradictory results.(Seidel et al. 1996) Using computational methods we have investigated the processes involved in the oxidation and reduction of DNA nucleobases.(Llano & Eriksson 2004a) In particular, the nature of the ET process, the effect of PT on such processes, and vice versa, was examined using the chemical models shown in **Figure 1**.

All optimizations were performed at the B3LYP/6–31(+)G(d,p) level of theory; diffuse functions only being included for anionic species'. Harmonic vibrational frequencies and zero–point vibrational energies (ZPVE) were calculated at this same level of theory. Relative energies and absolute chemical potentials were then obtained via single point calculations at the IEF-PCM/B3LYP/6–311+G(2df,p) level of theory, in aqueous solvent, based on the above optimized structures with inclusion of the appropriate Gibbs corrections. In combination with the values listed in **Table 1**, the free energies for oxidation of the four neutral DNA bases and the corresponding anions was determined. Specifically, the free energies were calculated for the three different reference states used to define the absolute chemical potential of the electron: in vacuum, aqueous or the SHE reference state.

the four DNA nucleobases, followed by adenine.(Hush & Cheung 1975; Kumar & Sevilla 2010; Steenken & Jovanovic 1997; Yang et al. 2004) Consequently, they are in general oxidized to give their radical-cations while thymine and cytosine act as electron sinks and form their radical-anions. However, vice versa, in the transfer of electron holes guanine typically acts as a sink for DNA radiative oxidation.(Cadet et al. 2008; Kumar & Sevilla 2010) Reduction/oxidation of a nucleobase can significantly affect its properties. In particular, it has been shown that their oxidation greatly increases their acidity (lowers their pKa).(Kumar & Sevilla 2010) Indeed, the formation of a radical base is often associated with proton transfer reactions that can lead to further nucleobase damage. However, as has also been noted, these same proton transfers can result in nucleobase repair.(Llano & Eriksson 2004b)

Fig. 1. Structures of the dehydrogenated nucleobases for both the cations and anions. The

chemical potential of the electron: in vacuum, aqueous or the SHE reference state.

Unfortunately, our understanding of these important electron- (ET) and proton-transfer (PT) reactions is incomplete.(Llano & Eriksson 2004a) This is not only due to the highly transient and reactive nature of key species involved, but also because experimental measurements of free energies for such processes can only be obtained indirectly.(Llano & Eriksson 2004a) Moreover, at times, the available experimental techniques (photoelectron spectroscopy, mass spectroscopy, pulse radiolysis and electrochemical techniques) appear to give contradictory results.(Seidel et al. 1996) Using computational methods we have investigated the processes involved in the oxidation and reduction of DNA nucleobases.(Llano & Eriksson 2004a) In particular, the nature of the ET process, the effect of PT on such processes, and vice versa, was examined using the chemical models shown in **Figure 1**. All optimizations were performed at the B3LYP/6–31(+)G(d,p) level of theory; diffuse functions only being included for anionic species'. Harmonic vibrational frequencies and zero–point vibrational energies (ZPVE) were calculated at this same level of theory. Relative energies and absolute chemical potentials were then obtained via single point calculations at the IEF-PCM/B3LYP/6–311+G(2df,p) level of theory, in aqueous solvent, based on the above optimized structures with inclusion of the appropriate Gibbs corrections. In combination with the values listed in **Table 1**, the free energies for oxidation of the four neutral DNA bases and the corresponding anions was determined. Specifically, the free energies were calculated for the three different reference states used to define the absolute

position of the missing hydrogen is marked by the asterisk.


Table 2. Calculated (see text) standard free energies (in kcal mol–1) of primary ionizations of the four nucleobases in aqueous solution. Experimental values are in parenthesis and taken from references: a (Seidel et al. 1996), b (Steenken et al. 1992), c (Steenken & Jovanovic 1997).

ET is a common process that occurs upon absorption of radiation by nucleobases. Using a first principles approach the free energy changes involved with such a process for all four DNA nucleobases were calculated and are shown in **Table 2**.(Llano & Eriksson 2002) It can be seen that the ionization of each of the anionic bases (B•–(aq)) can be either endothermic or exothermic depending on the reference state of the electron. For example, in the vacuum or aqueous state the oxidation of the anionic bases is generally an endothermic process. The only exception occurs for guanine in the aqueous state in which the process is marginally exothermic (–0.5 kcal mol–1). In contrast, in the case of the SHE reference state, oxidation of each base is markedly exothermic. For A•– and G•– the values calculated are in close agreement to those obtained experimentally.(Seidel et al. 1996; Steenken & Jovanovic 1997; Steenken et al. 1992) In contrast, those calculated for C•– and T•– are not in as good agreement, being almost twice the corresponding experimental values. However, the overall trends are consistent; the oxidation of C•– or T•– is thermodynamically less favorable than that of A•– or G•–. Conversely, the reverse process, capture of a solvated electron by C or T (i.e., C/T(aq) + e– (aq) C/T•–(aq)) is thermodynamically preferred (–9.9 kcal mol–1 and –13.5 kcal mol–1, respectively) compared to that involving A or G (–2.3 kcal mol–1 and 0.5 kcal mol–1, respectively).

Oxidation of the neutral bases is calculated to be endothermic for each reference state of the electron (**Table 2**). The degree of endothermicity, however, depends on the reference state being most endothermic in vacuum and least for the SHE reference state. Unlike that observed for the radical anion bases, the SHE calculated values for the neutral bases are all in good agreement with experiment. The largest difference occurs for C and is now only 10.7 kcal mol–1 compared to the 25.9 kcal mol–1 difference for C•–, while the smallest difference (– 3.8 kcal mol–1) is observed for guanine. In addition, neutral G is calculated to have the lowest free energy of oxidation and is thus the easiest of the four DNA nucleobases to be ionized, in agreement with experimental observations.

The free energies associated with the loss of a proton from the resulting radical-cationic bases to solution were calculated and are given in **Table 3**. For A/G•+ the energy changes associated with deprotonations to form *syn*–A(N6–H), *anti*–A(N6–H), and G(N1–H) were determined to be quite small at just –0.6, –0.7 and –0.3 kcal mol–1 respectively.(Llano & Eriksson 2004a) In contrast, the energy changes associated with deprotonation of C/T•+ to give *syn–*C(N4–H) or *anti–*C(N4–H) and T(C5–H) are larger at –5.2, –4.5 and –22.3 kcal mol–1, respectively.(Llano & Eriksson 2004a) It is noted that T(C5–H) has only been observed in the solid state and not in solution,(Steenken 1989) and thus will not be discussed herein. Unlike the other deprotonation processes, formation of T(N3–H) was found to be endothermic and

Mechanisms of Mutagenic DNA Nucleobase Damages and

+e–

(Steenken & Jovanovic 1997).

+ MeS•(aq).

Their Chemical and Enzymatic Repairs Investigated by Quantum Chemical Methods 395

Under alkaline conditions it is expected that ET would occur prior to PT, as seen in **Table 4**. However, under acidic conditions a PTET process may occur. The associated calculated energy values are given in **Table 5**. As can be seen, the free energy changes for ET are exothermic for all reference states when it occurs simultaneously with a PT. Importantly, the free energies associated with electron addition are decidedly more favorable in the case of a

B(–H)•(aq) B(aq)

The aqueous state reduction of the neutral radical bases (B(–H)•(aq)) by addition of H•(aq) was calculated to be exothermic for all DNA bases. More specifically, the free energy changes for reduction of *syn / anti*–A(N6–H)• and *–*C(N4–H)• are –85.0, –85.1, –91.4 and –92.0 kcal mol–1, respectively while for G(N1–H)• and T(N3–H)• they are –74.6 and –104.1 kcal mol–1, respectively. It is likely that this process is important under low pH conditions where the free protons would scavenge the hydrated electrons to yield H•. These free energy changes

under acidic conditions the PTET and H• processes would likely compete as they both react at diffusion-limited rates. Of all possible three reduction mechanisms for B(–H)•, PTET

> Deprotonated Radical Base ∆ETG<sup>Θ</sup> ∆PTETG<sup>Θ</sup> *syn*–A(N6–H)• –5.1 –13.5 *anti*–A(N6–H)• –5.8 –13.6 G(N1–H)• –3.4 –3.1 T(N3–H)• –32.6 –32.6 *syn–*C(N4–H)• –14.9 –19.9 *anti–*C(N4–H)• –15.3 –20.5

Table 6. Calculated (see text) standard Gibbs energies (in kcal mol–1) of the regeneration of the nucleobases by methylthiol. ∆ETGΘ is the free energy change for B(–H)•(aq) + MeS–

B(–H)–(aq) + MeS•(aq) while ∆PTETGΘ is the free energy change for B(–H) •(aq) + MeSH(aq) = B(aq)

Clearly, the possible processes by which the neutral bases may be regenerated are inherently favorable regardless of whether the electrons originate from vacuum, dilute aqueous

are less exothermic than those seen in the PTET process (cf. **Table 5**; e–

processes show the greatest change in free energy.

*syn*–A(N6–H)• –136.0 –97.6 –36.0 (–46.8)a A *anti*–A(N6–H)• –136.1 –97.7 –36.1 (–46.8)a A G(N1–H)• –125.6 –87.3 –26.5 (–19.7)a G T(N3–H)• –155.1 –116.7 –55.1 T *syn–*C(N4–H)• –142.4 –104.0 –42.4 C *anti–*C(N4–H)• –143.0 –104.6 –43.0 C Table 5. Calculated (see text) standard Gibbs energies of the regeneration of the nucleobases via a PTET process. Experimental values included in parenthesis taken from references: a

(aq) +e–

(SHE)

(aq) results). However,

(aq) =

PTET process compared to that observed in the previous ET process (cf. **Table 4**).

(vac) +e–


Table 3. Calculated (see text) standard Gibbs energies (in kcal mol–1) of the decay of the radical cations. Experimental values included in parenthesis taken from references: a (Steenken 1989; Steenken 1992).

had the largest absolute free energy change (9.9 kcal mol–1).(Llano & Eriksson 2004a) Thus, the loss of a proton in aqueous conditions is thermodynamically favorable for the radicalcationic bases except for proton loss from N3—H in thymine. The unfavourable decay of T•+ via deprotonation of N3–H suggests that the ion may have a long enough lifetime such that it may instead obtain an electron, a thermodynamically favourable process (**Table 2**). Thus, T•+ may instead preferably react to regenerate the neutral base T rather than decay.(Llano & Eriksson 2004a)

Having established an understanding of the energies associated with oxidation of the anionic and neutral bases, energy changes associated with possible repair mechanisms of this damage were then investigated. For the deprotonated neutral radical bases (B(–H)•(aq)) regeneration can occur via three possible pathways: (i) ET followed by PT (**Table 4**); (ii) a proton-coupled electron transfer (PTET) (**Table 5**) or (iii) direct transfer of a hydrogen atom. From **Table 4** it can be seen that reduction of the deprotonated bases is exothermic under all reference states with the exception of T(N3–H)• in the SHE reference state. However, the energy change for protonating all of the reduced bases (B(–H)– (aq)) is favorable. Protonation of G(N1–H)— has the smallest free energy change suggesting that the conjugate acid is relatively stronger than the other neutral bases. Importantly, the more acidic a molecule, the more powerful it is as a reducing agent. Thus, as G has the smallest energy cost of deprotonation of any of the DNA bases it is more likely to reduce the other bases and thereby act as an electron hole sink, as observed experimentally.


Table 4. Calculated (see text) standard Gibbs energies (in kcal mol–1) of the regeneration of the nucleobases via a ET then PT process. Experimental values included in parenthesis taken from references: a (Steenken 1989; Steenken 1992).

Cationic Radical Base ∆G (kcal mol–1) Deprotonated Radical Base A•+ –0.7 (≤ 1.4)a *syn*–A(N6–H), A•+ –0.6 (≤ 1.4)a *anti*–A(N6–H) G•+ –0.3 (5.3)a G(N1–H) T•+ –22.3 T(C5–H) T•+ 9.9 (4.9)a T(N3–H) C•+ –5.2 (~5.4)a *syn–*C(N4–H), C•+ –4.5 (~5.4)a *anti–*C(N4–H)

Table 3. Calculated (see text) standard Gibbs energies (in kcal mol–1) of the decay of the radical cations. Experimental values included in parenthesis taken from references: a

had the largest absolute free energy change (9.9 kcal mol–1).(Llano & Eriksson 2004a) Thus, the loss of a proton in aqueous conditions is thermodynamically favorable for the radicalcationic bases except for proton loss from N3—H in thymine. The unfavourable decay of T•+ via deprotonation of N3–H suggests that the ion may have a long enough lifetime such that it may instead obtain an electron, a thermodynamically favourable process (**Table 2**). Thus, T•+ may instead preferably react to regenerate the neutral base T rather than decay.(Llano &

Having established an understanding of the energies associated with oxidation of the anionic and neutral bases, energy changes associated with possible repair mechanisms of this damage were then investigated. For the deprotonated neutral radical bases (B(–H)•(aq)) regeneration can occur via three possible pathways: (i) ET followed by PT (**Table 4**); (ii) a proton-coupled electron transfer (PTET) (**Table 5**) or (iii) direct transfer of a hydrogen atom. From **Table 4** it can be seen that reduction of the deprotonated bases is exothermic under all reference states with the exception of T(N3–H)• in the SHE reference state. However, the

of G(N1–H)— has the smallest free energy change suggesting that the conjugate acid is relatively stronger than the other neutral bases. Importantly, the more acidic a molecule, the more powerful it is as a reducing agent. Thus, as G has the smallest energy cost of deprotonation of any of the DNA bases it is more likely to reduce the other bases and

B(–H)•(aq) B(–H)–(aq) B(aq)

*syn*–A(N6–H)• –113.2 –74.9 –13.2 *syn*–A(N6–H)– –22.7 (≥ –19.1)a A *anti*–A(N6–H)• –113.9 –75.6 –13.9 *anti*–A(N6–H)– –22.2 (≥ –19.1)a A G(N1–H)• –111.5 –73.2 –11.5 G(N1–H)– –14.4 (–13.0)a G T(N3–H)• –72.3 –34.0 27.7 T(N3–H)– –50.6 T *syn–*C(N4–H)• –123.0 –84.6 –23.0 *syn–*C(N4–H)– –19.4 (–17.7)a C *anti–*C(N4–H)• –123.4 –85.0 –23.4 *anti–*C(N4–H)– –19.6 (–17.7)a C Table 4. Calculated (see text) standard Gibbs energies (in kcal mol–1) of the regeneration of the nucleobases via a ET then PT process. Experimental values included in parenthesis taken

(SHE) +H+(aq)

(aq)) is favorable. Protonation

energy change for protonating all of the reduced bases (B(–H)–

thereby act as an electron hole sink, as observed experimentally.

(aq) +e–

(vac) +e–

from references: a (Steenken 1989; Steenken 1992).

(Steenken 1989; Steenken 1992).

+e–

Eriksson 2004a)

Under alkaline conditions it is expected that ET would occur prior to PT, as seen in **Table 4**. However, under acidic conditions a PTET process may occur. The associated calculated energy values are given in **Table 5**. As can be seen, the free energy changes for ET are exothermic for all reference states when it occurs simultaneously with a PT. Importantly, the free energies associated with electron addition are decidedly more favorable in the case of a PTET process compared to that observed in the previous ET process (cf. **Table 4**).


Table 5. Calculated (see text) standard Gibbs energies of the regeneration of the nucleobases via a PTET process. Experimental values included in parenthesis taken from references: a (Steenken & Jovanovic 1997).

The aqueous state reduction of the neutral radical bases (B(–H)•(aq)) by addition of H•(aq) was calculated to be exothermic for all DNA bases. More specifically, the free energy changes for reduction of *syn / anti*–A(N6–H)• and *–*C(N4–H)• are –85.0, –85.1, –91.4 and –92.0 kcal mol–1, respectively while for G(N1–H)• and T(N3–H)• they are –74.6 and –104.1 kcal mol–1, respectively. It is likely that this process is important under low pH conditions where the free protons would scavenge the hydrated electrons to yield H•. These free energy changes are less exothermic than those seen in the PTET process (cf. **Table 5**; e– (aq) results). However, under acidic conditions the PTET and H• processes would likely compete as they both react at diffusion-limited rates. Of all possible three reduction mechanisms for B(–H)•, PTET processes show the greatest change in free energy.


Table 6. Calculated (see text) standard Gibbs energies (in kcal mol–1) of the regeneration of the nucleobases by methylthiol. ∆ETGΘ is the free energy change for B(–H)•(aq) + MeS– (aq) = B(–H)–(aq) + MeS•(aq) while ∆PTETGΘ is the free energy change for B(–H) •(aq) + MeSH(aq) = B(aq) + MeS•(aq).

Clearly, the possible processes by which the neutral bases may be regenerated are inherently favorable regardless of whether the electrons originate from vacuum, dilute aqueous

Mechanisms of Mutagenic DNA Nucleobase Damages and

subsequent oxidation to generate the 8-oxo purine derivative.

Eriksson 2004b)

inclusion of the appropriate Gibbs corrections.

adenine and guanine respectively.

Their Chemical and Enzymatic Repairs Investigated by Quantum Chemical Methods 397

Fig. 2. Two proposed pathways for •OH attack at the C8 position of a purine base and

The **8–oxoG/A** products are in fact major stable DNA purine oxidation products produced via the radiolysis of water.(Hagen 1986; Hatahet 1998; Kasai et al. 1986; Teoule 1987) However, it is been observed that the adenine derivative (**8-oxoA**) is formed only about one–third of the time compared to the guanine derivative (**8-oxoG**).(Burrows & Muller 1998; Llano & Eriksson 2004b) Importantly, however, the formation of either **8–oxoB** species promotes the formation of mutagenic lesions that can cause mispairings resulting in G:C and T:A transversions.(Cheng et al. 1992; Cullis et al. 1996; Llano & Eriksson 2004b; Newcomb 1998; Pavlov et al. 1994) Such lesions are in fact caused by the further oxidation of **8–oxoB** to their corresponding **8–oxoB•(–H7)** derivatives. Moreover, it has been shown that depending on the reaction conditions both spiroiminodi- and guanidino-hydantoin are also major products formed by further oxidation of **8-oxoG**.(Munk et al. 2008) It has been suggested that these further reactions occur because the **8–oxoB** species' have lower ionization potentials than any other native base.(Cadet & Vigny 1990; Chatgilialoglu et al. 2000) Hence, they could act as a trap for a migrating electron hole.(Sponer et al. 2004; Yao et al. 2005) Unfortunately, however, despite detailed experimental study, the exact route by which such species' (**8-oxoB** and **8–oxoB•(–H7)**) may be formed remains unclear.(Llano &

We performed a computational investigation on the processes by which **8–OHB(aq)** may be formed for purine, adenine and guanine.(Llano & Eriksson 2004b) In addition, the subsequent oxidations by which **8–oxoB** and **8–oxoB•(–H7)** may be formed were also examined using a first principles approach.(Llano & Eriksson 2002) Optimized structures, harmonic vibrational frequencies and ZPVEs were calculated at the B3LYP/6–31(+)G(d,p) level theory; diffuse functions only being included for anionic species'. Relative Gibbs Free energies were obtained by performing single point calculations at the IEF–PCM/B3LYP/6– 311+G(2df,p) level of theory, in aqueous solvent, based on the above structures with

Attack of •OH at the C8 position in purine, adenine or guanine results in formation of the corresponding radical hydroxylated intermediates **B8OH•** (**Figure 3**). Notably, they are stabilized relative to the isolated reactants by –13.4, –14.9 and –15.8 kcal mol–1 for purine,

The oxidation of **B8OH•(aq)** may then be initiated via PT from its –C8—H moiety (**Figure 3**). This step, resulting in formation of the corresponding radical-anionic derivatives **8–OHB•–(aq)** was calculated to be significantly endothermic for all three purine bases considered with a minimum free energy cost of ~28 kcal mol–1. Notably, the largest costs of 35.4 and 47.4 kcal mol–1 are observed for adenine and guanine, respectively. This suggests that a strong base is

solutions or SHE reference states. However, they still require that there be a suitable reductant. It has been noted that in solution a free thiol could be a likely reductant for transfer of a H• to a radical-cationic DNA base, thereby repairing the lesion.(von Sonntag 1987; von Sonntag 1996). Hence, the applicability of thiols to act as such a reductant was also investigated. It should be noted that although the process of repairing the nucleobase can occur via enzymatic or other chemical processes, only the latter involving methylthiol is discussed here. The calculated free energy costs associated with regeneration of the deprotonated radical bases by methylthiol (CH3SH) are given in **Table 6**.

Two possible reduction pathways exist:


**Pathway i** most likely only occurs under basic conditions. However, as can be seen from **Table 6**, for *syn*– / *anti*–A(N6–H)• and *–*C(N4–H)• an initial loss of the –SH proton markedly lowers the reductive ability of the thiol. That is, the overall free energy change associated with regeneration of the nucleobases is reduced, i.e., ∆ETGΘ is less exothermic than ∆PTETGΘ. However, for G(N1–H)• and T(N3–H)• the free energy changes are quite close for both possible regeneration pathways. The markedly larger values of ∆PTETGΘ compared to ∆ETG<sup>Θ</sup> for all adenine and cytosine species considered suggests that PTET is the favoured process at any pH for these nucleobases. However, the negligible difference observed for guanine and thymine suggest that the preferred path will depend on the reaction conditions, e.g., pH. Importantly, regardless of the preferred pathway the calculated free energies indicate they are both favorable, exothermic processes.

### **3. 8-Oxopurine formation in purine, adenine and guanine**

A major type of secondary DNA damage is that caused by the attack of hydroxyl radicals.(Llano & Eriksson 2004a; von Sonntag 1987; von Sonntag 1996) Such radicals can be formed when metals or hydrogen peroxide are present.(Burrows & Muller 1998) In addition, however, the absorption of radiation by water can lead to not only the formation of solvated electrons but also of reactive oxygen species such as •OH. Furthermore, related modifications of nucleobases can occur via the reaction of their radical cation with water. For instance, reaction of G•+ with H2O has been suggested to be an important alternative pathway in DNA modification.(Candeias & Steenken 2000) Unfortunately, due to the high reactivity of these radicals where the rates of reaction are typically diffusion controlled,(Llano & Eriksson 2004a) it is impossible for radical scavengers to prevent them from damaging DNA. It has been suggested that approximately half of the damage done by •OH occurs at the nucleobases. In the purine bases the hydroxyl has been observed to attack at their double bonds to form the C4, C5 and C8 adducts with the latter (i.e., **B8OH•(aq)**) being the major product of oxidation and radiolysis.(Hagen 1986; Hatahet 1998; Kasai et al. 1986; Llano & Eriksson 2004b; Teoule 1987)

The subsequent oxidation of **B8OH•(aq)** to give **8–OHB(aq)** may occur via either of the two pathways shown in **Figure 2**: (i) a PT followed by an ET(Cullis et al. 1996) or (ii) a PTET mechanism(Candeias & Steenken 2000). The resulting common **8–OHB(aq)** product of both pathways can then undergo tautomerization to yield the corresponding 8–oxo derivative (**8– oxoB(aq)**). It is noted that the barrier for tautomerization of 8–hydroxy–purine (**8–OHPu(aq)**) has been calculated to be quite low at only 7.0 kcal mol–1 with the keto form (**8–oxoPu(aq)**) being favoured over the enol form by 10.6 kcal mol–1. (Llano & Eriksson 2004b)

solutions or SHE reference states. However, they still require that there be a suitable reductant. It has been noted that in solution a free thiol could be a likely reductant for transfer of a H• to a radical-cationic DNA base, thereby repairing the lesion.(von Sonntag 1987; von Sonntag 1996). Hence, the applicability of thiols to act as such a reductant was also investigated. It should be noted that although the process of repairing the nucleobase can occur via enzymatic or other chemical processes, only the latter involving methylthiol is discussed here. The calculated free energy costs associated with regeneration of the

**Pathway i** most likely only occurs under basic conditions. However, as can be seen from **Table 6**, for *syn*– / *anti*–A(N6–H)• and *–*C(N4–H)• an initial loss of the –SH proton markedly lowers the reductive ability of the thiol. That is, the overall free energy change associated with regeneration of the nucleobases is reduced, i.e., ∆ETGΘ is less exothermic than ∆PTETGΘ. However, for G(N1–H)• and T(N3–H)• the free energy changes are quite close for both possible regeneration pathways. The markedly larger values of ∆PTETGΘ compared to ∆ETG<sup>Θ</sup> for all adenine and cytosine species considered suggests that PTET is the favoured process at any pH for these nucleobases. However, the negligible difference observed for guanine and thymine suggest that the preferred path will depend on the reaction conditions, e.g., pH. Importantly, regardless of the preferred pathway the calculated free energies indicate

A major type of secondary DNA damage is that caused by the attack of hydroxyl radicals.(Llano & Eriksson 2004a; von Sonntag 1987; von Sonntag 1996) Such radicals can be formed when metals or hydrogen peroxide are present.(Burrows & Muller 1998) In addition, however, the absorption of radiation by water can lead to not only the formation of solvated electrons but also of reactive oxygen species such as •OH. Furthermore, related modifications of nucleobases can occur via the reaction of their radical cation with water. For instance, reaction of G•+ with H2O has been suggested to be an important alternative pathway in DNA modification.(Candeias & Steenken 2000) Unfortunately, due to the high reactivity of these radicals where the rates of reaction are typically diffusion controlled,(Llano & Eriksson 2004a) it is impossible for radical scavengers to prevent them from damaging DNA. It has been suggested that approximately half of the damage done by •OH occurs at the nucleobases. In the purine bases the hydroxyl has been observed to attack at their double bonds to form the C4, C5 and C8 adducts with the latter (i.e., **B8OH•(aq)**) being the major product of oxidation and radiolysis.(Hagen 1986; Hatahet 1998;

The subsequent oxidation of **B8OH•(aq)** to give **8–OHB(aq)** may occur via either of the two pathways shown in **Figure 2**: (i) a PT followed by an ET(Cullis et al. 1996) or (ii) a PTET mechanism(Candeias & Steenken 2000). The resulting common **8–OHB(aq)** product of both pathways can then undergo tautomerization to yield the corresponding 8–oxo derivative (**8– oxoB(aq)**). It is noted that the barrier for tautomerization of 8–hydroxy–purine (**8–OHPu(aq)**) has been calculated to be quite low at only 7.0 kcal mol–1 with the keto form (**8–oxoPu(aq)**)

being favoured over the enol form by 10.6 kcal mol–1. (Llano & Eriksson 2004b)

deprotonated radical bases by methylthiol (CH3SH) are given in **Table 6**.

**3. 8-Oxopurine formation in purine, adenine and guanine** 

i. B(–H)•(aq) + MeS—(aq) B(–H)—(aq) + MeS•(aq) ii. B(–H)•(aq) + MeSH(aq) B(aq) + MeS•(aq)

Two possible reduction pathways exist:

they are both favorable, exothermic processes.

Kasai et al. 1986; Llano & Eriksson 2004b; Teoule 1987)

Fig. 2. Two proposed pathways for •OH attack at the C8 position of a purine base and subsequent oxidation to generate the 8-oxo purine derivative.

The **8–oxoG/A** products are in fact major stable DNA purine oxidation products produced via the radiolysis of water.(Hagen 1986; Hatahet 1998; Kasai et al. 1986; Teoule 1987) However, it is been observed that the adenine derivative (**8-oxoA**) is formed only about one–third of the time compared to the guanine derivative (**8-oxoG**).(Burrows & Muller 1998; Llano & Eriksson 2004b) Importantly, however, the formation of either **8–oxoB** species promotes the formation of mutagenic lesions that can cause mispairings resulting in G:C and T:A transversions.(Cheng et al. 1992; Cullis et al. 1996; Llano & Eriksson 2004b; Newcomb 1998; Pavlov et al. 1994) Such lesions are in fact caused by the further oxidation of **8–oxoB** to their corresponding **8–oxoB•(–H7)** derivatives. Moreover, it has been shown that depending on the reaction conditions both spiroiminodi- and guanidino-hydantoin are also major products formed by further oxidation of **8-oxoG**.(Munk et al. 2008) It has been suggested that these further reactions occur because the **8–oxoB** species' have lower ionization potentials than any other native base.(Cadet & Vigny 1990; Chatgilialoglu et al. 2000) Hence, they could act as a trap for a migrating electron hole.(Sponer et al. 2004; Yao et al. 2005) Unfortunately, however, despite detailed experimental study, the exact route by which such species' (**8-oxoB** and **8–oxoB•(–H7)**) may be formed remains unclear.(Llano & Eriksson 2004b)

We performed a computational investigation on the processes by which **8–OHB(aq)** may be formed for purine, adenine and guanine.(Llano & Eriksson 2004b) In addition, the subsequent oxidations by which **8–oxoB** and **8–oxoB•(–H7)** may be formed were also examined using a first principles approach.(Llano & Eriksson 2002) Optimized structures, harmonic vibrational frequencies and ZPVEs were calculated at the B3LYP/6–31(+)G(d,p) level theory; diffuse functions only being included for anionic species'. Relative Gibbs Free energies were obtained by performing single point calculations at the IEF–PCM/B3LYP/6– 311+G(2df,p) level of theory, in aqueous solvent, based on the above structures with inclusion of the appropriate Gibbs corrections.

Attack of •OH at the C8 position in purine, adenine or guanine results in formation of the corresponding radical hydroxylated intermediates **B8OH•** (**Figure 3**). Notably, they are stabilized relative to the isolated reactants by –13.4, –14.9 and –15.8 kcal mol–1 for purine, adenine and guanine respectively.

The oxidation of **B8OH•(aq)** may then be initiated via PT from its –C8—H moiety (**Figure 3**). This step, resulting in formation of the corresponding radical-anionic derivatives **8–OHB•–(aq)** was calculated to be significantly endothermic for all three purine bases considered with a minimum free energy cost of ~28 kcal mol–1. Notably, the largest costs of 35.4 and 47.4 kcal mol–1 are observed for adenine and guanine, respectively. This suggests that a strong base is

Mechanisms of Mutagenic DNA Nucleobase Damages and

**B8OH•(aq)** for purine, adenine and guanine, respectively.

oxidation pathway in **B8OH•**.

**8–oxoB.**

Their Chemical and Enzymatic Repairs Investigated by Quantum Chemical Methods 399

exothermic for all bases considered by 10.0 – 12.3 kcal mol–1. It should be noted that in addition to the possible ET and PT initiated processes, a PTET (i.e., the concerted loss of electron and proton) process may also occur. However, the overall free energy change for formation of **8–OHB** via this alternate path is –27.9, –26.8 and –25.1 kcal mol–1 relative to

Fig. 4. Calculated (see text) standard Gibbs energies (kcal mol–1) for the ET initiated

The formation of **8–oxoB•(–H7)(aq)** can potentially be achieved by further oxidation of the neutral tautomers **8–oxoB(aq)** and **8–OHB(aq)**, initiated by loss of an electron (**Figure 5**). For both species', however, this ET process is endothermic. For purine and adenine the lowest associated energy costs (46.1 and 30.5 kcal mol–1 respectively) are incurred for ET from **8– oxoB(aq)** while for guanine it is incurred from **8–OHB(aq)** with a cost of 20.1 kcal mol–1. Notably, for both **8–oxoB(aq)** and **8–OHB(aq)** the loss of an electron from the guaninederivative (**8–oxoG(aq)** and **8–OHG(aq)**) is preferred to that from the corresponding adenine or purine-derivatives. Regardless, however, a suitable oxidant is required in order to oxidize either **8–oxoB(aq)** or **8–OHB(aq)**. The barriers for tautomerization for both the neutral **8– oxoB(aq)**/**8–OHB(aq)** and radical-cationic **8–OHB•+(aq)**/**8–oxoB•+(aq)** species' are quite low at

Fig. 5. Calculated (see text) standard Gibbs energies (kcal mol–1) for oxidation of **8–OHB** and

Fig. 3. Calculated (see text) standard Gibbs energies (kcal mol–1) for PT initiated oxidation of **B8OH•.** 

required for deprotonation of C8 in **B8OH•(aq)** and is unlikely to occur under neutral or acidic conditions. The resulting **8–OHB•–(aq)** ions can then either oxidize further via loss of an electron to give **8–OHB(aq)** or tautomerize to give **8–oxoB•–(aq)**. As can be seen in **Figure 3**, both possible processes are thermodynamically preferred. Importantly, however, these latter two species can subsequently undergo tautomerization and electron loss, respectively to give the same **8–oxoB(aq)** product species. The barriers for tautomerization of the radicalanionic (**8–OHPu•–(aq)**) and neutral purine (**8–OHPu(aq)**) are both low at just 5.2 and 7.1 kcal mol–1, respectively. (Llano & Eriksson 2004b) Thus, the choice of pathway from **8–OHB•–(aq)** to **8–oxoB(aq)** may in fact be controlled by the thermodynamics of the loss of an electron. From **Figure 3** it can be seen that for purine and guanine the largest decreases in free energy observed for electron loss along either of these two paths occurs for the oxidation of the enol radical anion **8–OHB•–(aq)** to give **8–OHB(aq)** with decreases of 55.6 and 72.5 kcal mol–1 respectively. In contrast, for adenine the largest decrease (65.2 kcal mol–1) is observed for loss of an electron from the keto radical anion **8–oxoB•–(aq)** to give **8–oxoB(aq)**. Thus for purine and guanine it is likely that formation of **8–oxoB(aq)** occurs via tautomerization of **8– OHB(aq)**, while for adenine it involves oxidation of **8–oxoB•–(aq)**.

As shown in **Figure 4**, oxidation of **B8OH•(aq)** may alternatively be initiated by ET, giving rise to **B8OH+(aq)** ions. Similar to that observed for an initial PT from **B8OH•(aq)** (cf. **Figure 3**), this process is also found to be endothermic for all three purine nucleobases. However, with the exception of purine, the energy costs incurred are now significantly less. Indeed, for **A/G8OH•(aq)** ET is thermodynamically preferred to PT by 24.1 and 47.2 kcal mol–1 respectively. It is further noted that for guanine, the reactant **G8OH•(aq)** is essentially thermoneutral with the **G8OH+(aq)** intermediate. Furthermore, the subsequent PT from **B8OH+(aq)** to give **8–OHB(aq)** is highly exothermic for all bases by 57.1, 45.3 and 25.3 kcal mol–1 for purine, adenine and guanine, respectively. This is in contrast to the highly endothermic initial PT observed in **B8OH•(aq)** (cf. **Figure 3**). For the ET initiated oxidation pathway tautomerization can only happen once PT has occurred, that is, once **8–OHB(aq)** has been formed. This rearrangement, which results directly in formation of **8–oxoB(aq)**, is

Fig. 3. Calculated (see text) standard Gibbs energies (kcal mol–1) for PT initiated oxidation of

required for deprotonation of C8 in **B8OH•(aq)** and is unlikely to occur under neutral or acidic conditions. The resulting **8–OHB•–(aq)** ions can then either oxidize further via loss of an electron to give **8–OHB(aq)** or tautomerize to give **8–oxoB•–(aq)**. As can be seen in **Figure 3**, both possible processes are thermodynamically preferred. Importantly, however, these latter two species can subsequently undergo tautomerization and electron loss, respectively to give the same **8–oxoB(aq)** product species. The barriers for tautomerization of the radicalanionic (**8–OHPu•–(aq)**) and neutral purine (**8–OHPu(aq)**) are both low at just 5.2 and 7.1 kcal mol–1, respectively. (Llano & Eriksson 2004b) Thus, the choice of pathway from **8–OHB•–(aq)** to **8–oxoB(aq)** may in fact be controlled by the thermodynamics of the loss of an electron. From **Figure 3** it can be seen that for purine and guanine the largest decreases in free energy observed for electron loss along either of these two paths occurs for the oxidation of the enol radical anion **8–OHB•–(aq)** to give **8–OHB(aq)** with decreases of 55.6 and 72.5 kcal mol–1 respectively. In contrast, for adenine the largest decrease (65.2 kcal mol–1) is observed for loss of an electron from the keto radical anion **8–oxoB•–(aq)** to give **8–oxoB(aq)**. Thus for purine and guanine it is likely that formation of **8–oxoB(aq)** occurs via tautomerization of **8–**

As shown in **Figure 4**, oxidation of **B8OH•(aq)** may alternatively be initiated by ET, giving rise to **B8OH+(aq)** ions. Similar to that observed for an initial PT from **B8OH•(aq)** (cf. **Figure 3**), this process is also found to be endothermic for all three purine nucleobases. However, with the exception of purine, the energy costs incurred are now significantly less. Indeed, for **A/G8OH•(aq)** ET is thermodynamically preferred to PT by 24.1 and 47.2 kcal mol–1 respectively. It is further noted that for guanine, the reactant **G8OH•(aq)** is essentially thermoneutral with the **G8OH+(aq)** intermediate. Furthermore, the subsequent PT from **B8OH+(aq)** to give **8–OHB(aq)** is highly exothermic for all bases by 57.1, 45.3 and 25.3 kcal mol–1 for purine, adenine and guanine, respectively. This is in contrast to the highly endothermic initial PT observed in **B8OH•(aq)** (cf. **Figure 3**). For the ET initiated oxidation pathway tautomerization can only happen once PT has occurred, that is, once **8–OHB(aq)** has been formed. This rearrangement, which results directly in formation of **8–oxoB(aq)**, is

**OHB(aq)**, while for adenine it involves oxidation of **8–oxoB•–(aq)**.

**B8OH•.** 

exothermic for all bases considered by 10.0 – 12.3 kcal mol–1. It should be noted that in addition to the possible ET and PT initiated processes, a PTET (i.e., the concerted loss of electron and proton) process may also occur. However, the overall free energy change for formation of **8–OHB** via this alternate path is –27.9, –26.8 and –25.1 kcal mol–1 relative to **B8OH•(aq)** for purine, adenine and guanine, respectively.

Fig. 4. Calculated (see text) standard Gibbs energies (kcal mol–1) for the ET initiated oxidation pathway in **B8OH•**.

The formation of **8–oxoB•(–H7)(aq)** can potentially be achieved by further oxidation of the neutral tautomers **8–oxoB(aq)** and **8–OHB(aq)**, initiated by loss of an electron (**Figure 5**). For both species', however, this ET process is endothermic. For purine and adenine the lowest associated energy costs (46.1 and 30.5 kcal mol–1 respectively) are incurred for ET from **8– oxoB(aq)** while for guanine it is incurred from **8–OHB(aq)** with a cost of 20.1 kcal mol–1. Notably, for both **8–oxoB(aq)** and **8–OHB(aq)** the loss of an electron from the guaninederivative (**8–oxoG(aq)** and **8–OHG(aq)**) is preferred to that from the corresponding adenine or purine-derivatives. Regardless, however, a suitable oxidant is required in order to oxidize either **8–oxoB(aq)** or **8–OHB(aq)**. The barriers for tautomerization for both the neutral **8– oxoB(aq)**/**8–OHB(aq)** and radical-cationic **8–OHB•+(aq)**/**8–oxoB•+(aq)** species' are quite low at

Fig. 5. Calculated (see text) standard Gibbs energies (kcal mol–1) for oxidation of **8–OHB** and **8–oxoB.**

Mechanisms of Mutagenic DNA Nucleobase Damages and

no delocalization onto the water itself.

**Table 7**.

Their Chemical and Enzymatic Repairs Investigated by Quantum Chemical Methods 401

neutral base,(Labet et al. 2008a) it forms a hydrogen-bond bridge between N3 and N4 (**RC**: **Figure 6**). Thus, only the *anti–* form is discussed herein. It is noted that the addition of H2O has negligible effect on the Mulliken charges and spin densities of *anti–*C(N4–H)• and with

Deamination is then initiated by transfer of a H• from the water onto the N3 ring centre (**Figure 6**). This process proceeds via **TS1** with a barrier of 16.8 kcal mol–1 to give intermediate **I1**, lying 11.5 kcal mol–1 higher in free energy than **RC** and is thus endergonic. Intermediate **I1** resembles a complex between a cytosine tautomer and a hydroxyl radical. Interestingly, upon H• transfer the partial charge on C4 has increased by +0.29e to 0.38e close to that observed on C4 (0.37e) in neutral cytosine. Thus, in **I1** C4 has the same electrophilicity as in the neutral base. Indeed, the next step is nucleophilic attack of the •OH moiety at the C4 centre. This occurs via **TS2** with a barrier of 8.6 kcal mol–1 relative to **I1**; 20.1 kcal mol–1 with respect to **RC**. It should be noted that for spontaneous deamination of cytosine in water the analogous hydroxylation also occurs via attack of water at the C4 position with simultaneous transfer of a proton from the H2O moiety onto the amino N4 centre with concomitant cleavage of the C4—N4 bond. However, it proceeds with a barrier four to five times larger than that observed above for reaction via **TS2**.(Labet et al. 2008b) In the resulting intermediate **I2**, lying just 4.2 kcal mol–1 higher in energy than the initial complex **RC**, the C4 centre is now tetrahedral with a C4—OH bond length of 1.432 Å. There are then five possible pathways by which deamination of **I2** may occur, hereafter referred to as **Path A**, **B**, **C**, **D**, and **E**. The free energy changes associated with each are summarized in

Fig. 6. Calculated (see text) relative free energies for initial abstraction of H• from H2O by

Path A ∆G Path B ∆G Path C ∆G Path D ∆G Path E ∆G **I2** 4.3 **I2** 4.3 **I2** 4.3 **I2** 4.3 **I2** 4.3 **AI3** –9.0 **BTS3** 38.3 **AI3** –9.0 **DTS3** 31.1 **AI3** –9.0 **AI4** –0.2 **BI3** 13.1 **CTS3** 30.4 **DI3** 6.5 **ETS3** 24.9 **ATS3** 7.1 **BI4** 7.5 **BI4** 7.5 **DI4** 0.1 **DI4** 0.1 **AP** –7.5 **BTS4** 9.4 **BTS4** 9.4 **DTS4** 2.2 **DTS4** 2.2

Table 7. Calculated (see text) relative free energies for stationary points along **Path A**, **B**, **C**,

**BP** 11.4 **BP** 11.4 **DP** –22.0 **DP** –22.0

*anti–*C(N4–H)• with subsequent attack of •OH at the C4 centre.

**D** and **E** for deamination of **I2** in aqueous solution.

just 7.0 and 9.2 kcal mol–1, respectively.(Llano & Eriksson 2004b) Thus, once formed, such species are expected to be able to easily interconvert between their enol and keto forms with equilibrium favouring the latter. However, once the **8–oxoB•+(aq)** species is formed, loss of a proton (H+) from N7 to give **8–oxoB•(–H7)(aq)** is exothermic for all bases (**Figure 5**). Notably, **8–oxoG•(–H7)(aq)** is calculated to lie 4.8 kcal mol–1 lower in energy than the corresponding adenine derivative **8–oxoA•(–H7)(aq)** and may thus help explain the preference of its formation over that of **8–oxoA**.
