**4. Deamination of oxidized cytosine**

In addition to damage by ionizing radiation it has been found that nucleobases are susceptible to oxidation by one–electron oxidants, e.g., nitrosoperoxycarbonate present during inflammatory processes.(Cadet et al. 2006; Lee et al. 2007) The purine base guanine, despite having the lowest ionization potential, is not the sole target for oxidants. In particular it has been observed that the pyrimidine bases are also susceptible to oxidation.(Decarroz et al. 1986; Douki & Cadet 1999; Wagner et al. 1990) Importantly, this oxidation leads to degradation via two possible competing pathways that are initiated by deprotonation of: (i) the methylene carbon of the sugar moiety (C1') attached to the pyrimidine nitrogen (N1) or (ii) the exocyclic amine attached to C4 of the ring. Notably, the latter path has been suggested to lead to hydrolytic deamination of the pyrimidine ring.(Decarroz et al. 1987)

Previous computational investigations have investigated the deamination of non-oxidized cytosine, in particular via the attack of water or an hydroxyl anion (–OH) at its C4 centre and via NO• attack at N4.(Almatarneh et al. 2006; Labet et al. 2009) While the lowest barrier was obtained for the nucleophilic attack of OH– at C4, the calculated value was 9.6 kcal mol–1 higher than that obtained experimentally. However, the susceptibility of cytosine to one– electron oxidation suggests that possible mechanisms for deamination of C•+ should also be taken into consideration. The importance of considering such reactions is further underlined by the fact that the product of oxidation and deamination of cytosine is the highly mutagenic uracil residue.

We used computational chemistry methods to investigate the deamination of cytosine via the oxidized cytosine intermediate C•+ and via a deprotonated cytosine. Optimized structures and their corresponding harmonic vibrational frequencies were obtained at the IEF–PCM/B3LYP/6–311G(d,p) level of theory in aqueous solvent. Relative free energies were obtained at the same level of theory with inclusion of the appropriate Gibbs energy corrections.

In **section 2** it was shown that the one–electron oxidation of cytosine with the loss of the electron and proton in aqueous solution (i.e. C e– (aq) + H+(aq) + C(–N4)•(aq)) occurs with a sizeable free energy cost of approximately 104 kcal mol–1 (**Table 4**). The optimized structure of the oxidized C(–N4)•(aq) ring (not shown) is similar to that of neutral cytosine being planar with similar bond lengths, in agreement with other theoretical studies.(Cauet et al. 2006; Wetmore et al. 2000; Wetmore et al. 1998) The calculated spin densities and Mulliken charges showed that the positive charge is delocalized over the ring with the greatest change in partial charges occurring at C5 (+0.24e) while for spin densities they occur at C5 (0.64) and N1 (0.30).

The loss of a proton from N4 in C•+ can result in the formation of either *syn–* or *anti–*C(N4– H)• with the former being slightly more stable (**Table 4**). However, in the resulting *anti–* form it was found that when H2O is added, analogous to spontaneous deamination of the

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

In addition to damage by ionizing radiation it has been found that nucleobases are susceptible to oxidation by one–electron oxidants, e.g., nitrosoperoxycarbonate present during inflammatory processes.(Cadet et al. 2006; Lee et al. 2007) The purine base guanine, despite having the lowest ionization potential, is not the sole target for oxidants. In particular it has been observed that the pyrimidine bases are also susceptible to oxidation.(Decarroz et al. 1986; Douki & Cadet 1999; Wagner et al. 1990) Importantly, this oxidation leads to degradation via two possible competing pathways that are initiated by deprotonation of: (i) the methylene carbon of the sugar moiety (C1') attached to the pyrimidine nitrogen (N1) or (ii) the exocyclic amine attached to C4 of the ring. Notably, the latter path has been suggested to lead to

Previous computational investigations have investigated the deamination of non-oxidized cytosine, in particular via the attack of water or an hydroxyl anion (–OH) at its C4 centre and via NO• attack at N4.(Almatarneh et al. 2006; Labet et al. 2009) While the lowest barrier was obtained for the nucleophilic attack of OH– at C4, the calculated value was 9.6 kcal mol–1 higher than that obtained experimentally. However, the susceptibility of cytosine to one– electron oxidation suggests that possible mechanisms for deamination of C•+ should also be taken into consideration. The importance of considering such reactions is further underlined by the fact that the product of oxidation and deamination of cytosine is the highly

We used computational chemistry methods to investigate the deamination of cytosine via the oxidized cytosine intermediate C•+ and via a deprotonated cytosine. Optimized structures and their corresponding harmonic vibrational frequencies were obtained at the IEF–PCM/B3LYP/6–311G(d,p) level of theory in aqueous solvent. Relative free energies were obtained at the same level of theory with inclusion of the appropriate Gibbs energy

In **section 2** it was shown that the one–electron oxidation of cytosine with the loss of the

sizeable free energy cost of approximately 104 kcal mol–1 (**Table 4**). The optimized structure of the oxidized C(–N4)•(aq) ring (not shown) is similar to that of neutral cytosine being planar with similar bond lengths, in agreement with other theoretical studies.(Cauet et al. 2006; Wetmore et al. 2000; Wetmore et al. 1998) The calculated spin densities and Mulliken charges showed that the positive charge is delocalized over the ring with the greatest change in partial charges occurring at C5 (+0.24e) while for spin densities they occur at C5

The loss of a proton from N4 in C•+ can result in the formation of either *syn–* or *anti–*C(N4– H)• with the former being slightly more stable (**Table 4**). However, in the resulting *anti–* form it was found that when H2O is added, analogous to spontaneous deamination of the

(aq) + H+(aq) + C(–N4)•(aq)) occurs with a

formation over that of **8–oxoA**.

mutagenic uracil residue.

corrections.

(0.64) and N1 (0.30).

**4. Deamination of oxidized cytosine** 

hydrolytic deamination of the pyrimidine ring.(Decarroz et al. 1987)

electron and proton in aqueous solution (i.e. C e–

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 no delocalization onto the water itself.

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 **Table 7**.

Fig. 6. Calculated (see text) relative free energies for initial abstraction of H• from H2O by *anti–*C(N4–H)• with subsequent attack of •OH at the C4 centre.


Table 7. Calculated (see text) relative free energies for stationary points along **Path A**, **B**, **C**, **D** and **E** for deamination of **I2** in aqueous solution.

Mechanisms of Mutagenic DNA Nucleobase Damages and

al. 2004)

involved.

Their Chemical and Enzymatic Repairs Investigated by Quantum Chemical Methods 403

thermodynamically favoured. In contrast, in those cases in which only one proton originates from solution (**Paths B – E**), only those involving intramolecular rearrangement of a proton from C4—OH (**Paths D** and **E**) are exothermic and lead to formation of the most thermodynamically preferred product (**DP**). Furthermore, the barrier for this rearrangement is lowest when it occurs prior to proton donation from the solvent. Lastly in the case where both protonations occur via intermolecular processes, NH3•+ is formed (**AP**) while NH3 is

As mentioned in **section 2**, the exposure of DNA to high–energy radiation can also cause damage at its phosphate backbone. In particular, it can cause strand breaks via cleavage of the phosphoester bonds.(Lipfert et al. 2004) When the absorption of radiation causes the ionization of the phosphate it has been shown that it then abstracts a H• from the deoxyribose ring at either its C4' or C5' position. This is then followed by heterolytic cleavage of the phosphoester bond.(Lipfert et al. 2004; Steenken & Goldbergerova 1998) Alternatively, the strand break may be preceded by chemical modification of the nucleobase or deoxyribose ring or the phosphoester bond may simply undergo a direct cleavage.(Lipfert et

Experimentally it can be difficult to clearly observe and characterize damage within DNA due to its size. Thus, it is common to either use short fragments or model compounds that can reproduce or mimic the damage and associated processes that may occur. For example, serine phosphate contains a phosphate bond as well as a carboxylate that can act as an electron scavenger much like the bases within DNA itself. Thus, it is often used in experimental studies on the processes of DNA damage at its phosphate and subsequent bond cleavage reactions and in fact has led to a deeper understanding of those processes

(**I**) (**II**)(**III**)

phosphate. (Sanderud & Sagstuen 1996)

Fig. 7. The C2-centered radical **I** and C3-centered radicals **II** and **III** proposed to be formed upon the irradiation of non– and partially-deuterated single crystals of L–*O*–serine

In particular, evidence has been obtained suggesting the formation of several different radical species. Specifically, the irradiation of non– and partially-deuterated single crystals of L–*O*–serine phosphate has been suggested to produce the three radicals **I, II** and **III** shown in **Figure 7**.(Sanderud & Sagstuen 1996) In particular, it is thought that upon irradiation serine phosphate can take up a now free electron to form a C1-centered radical anion. This may then undergo deamination to form the C2-centered radical anion **I**. Alternatively, a serine phosphate may lose an electron to form a neutral C1-centered radical which then undergoes decarboxylation to give a C2-centered radical. This latter radical may then abstract a H• from the C3 position of another serine phosphate to generate the C3 centered radical anion **II**. In contrast, the neutral C3-centered radical **III** is proposed to be

formed when only a single proton originates from exogenous sources (**BP** and **DP**).

**5. Oxidation of serine phosphate: Implications for DNA** 

**Path A** begins with protonation of the N4 centre in **I2** where the proton originates from the surrounding dilute aqueous solvent. The resulting intermediate **AI3** lies 13.3 kcal mol–1 lower in energy than **I2**. This is followed by a second protonation of N4 with the proton again originating from the surrounding dilute aqueous solvent. In contrast to the first, this second protonation is endergonic by 8.8 kcal mol–1. However, the resulting intermediate **AI4** still lies lower in energy than **I2** by 4.5 kcal mol–1. In **AI4** the initial –N4H group in **I2** has now formally become an –N4H3+ group. Consequently, the next step is the loss of NH3 by simple cleavage of the C—N bond. This occurs with a barrier of 7.3 kcal mol–1 relative to **AI4**  (**Table 7**). It should be noted that the resulting product complex of **AP** lies 11.8 kcal mol–1 lower in energy than **I2** and resembles a complex between NH3•+ and protonated uracil.(Labet et al. 2008b)

In contrast, in **Path B** the initial 'protonation' of N4 is achieved via an intramolecular rearrangement from the N3—H group and does not originate from solution. This reaction proceeds via the four-membered ring TS **BTS3** at a significant cost of 34.0 kcal mol–1 to give intermediate **BI3**, lying 8.8 kcal mol–1 higher in energy than **I2** (**Table 7**). However, similar to **Path A** the second step is protonation of N4 with the proton originating from solution. This process is exergonic with the resulting intermediate **BI4** lying 5.6 kcal mol–1 lower in free energy than **BI3**. The subsequent cleavage of the C4—N4H3+ bond occurs via **BTS4** to give the product complex **BP**. However, unlike for **Path A**, **BP** is higher in energy than **I2** by 7.1 kcal mol–1 and instead resembles a complex between NH3 and the oxidized enol form of Uracil. Like **Path A**, the alternate **Path C** begins with the exothermic protonation of N4 by a proton originating from solution to give **AI3** (**Table 7**). Now, however, the second proton is obtained via the intramolecular proton transfer that initiated **Path B**, i.e., by transfer from the N3—H group. This reaction proceeds via **CTS3** with a considerable barrier of 39.4 kcal mol–1, even higher than that observed in **Path B**, to give intermediate **BI4**. The remaining step, cleavage of the C4—N4 bond, is then identical to that of **Path B** as is the product formed.

**Path D**, unlike the previous paths, involves an initial intramolecular proton transfer from the C4—OH hydroxyl onto the N4 centre (**Table 7**). This reaction proceeds via **DTS3** at a cost of 26.8 kcal mol–1. While this barrier is still quite high, it is 7.2 and 12.6 kcal mol–1 lower than the analogous intramolecular rearrangements in **Paths B** and **C** and is thus kinetically favoured. The resulting intermediate formed **DI3** lies only 2.2 kcal mol–1 higher in energy than **I2**. This is then followed by protonation of the N4 centre by a proton originating from solution, similar to **Paths A** and **B**. This step is exothermic with the intermediate formed, **DI4**, lying 6.4 kcal mol–1 lower in energy than **DI3**. The next and final step is the loss of NH3 and occurs via **DTS4** with a quite small barrier of just 2.1 kcal mol–1. The resulting product **DP**, resembling a complex between NH3 and oxidized uracil, is markedly lower in energy than **I2** by 26.3 kcal mol–1. Importantly, it is in fact the lowest energy product complex obtained of all those considered herein.

The final path considered, **Path E**, begins with the same exothermic intermolecular protonation of N4 as in **Paths A** and **C** and again leads to formation of **AI3**. The next step, however, is intramolecular proton transfer from the C4—OH group to N4 as occurs in **Path D**. Importantly, it proceeds via **ETS3** with a higher barrier (33.9 kcal mol–1) than observed in **Path D** (26.8 kcal mol–1) to give **DI4**. The final step is then cleavage of the C4—N4 bond as in **Path D** to give **DP**.

Thus, while deamination via donation of both protons from the aqueous solution (**Path A**) may have the lowest associated barriers, the resulting product is not the most

**Path A** begins with protonation of the N4 centre in **I2** where the proton originates from the surrounding dilute aqueous solvent. The resulting intermediate **AI3** lies 13.3 kcal mol–1 lower in energy than **I2**. This is followed by a second protonation of N4 with the proton again originating from the surrounding dilute aqueous solvent. In contrast to the first, this second protonation is endergonic by 8.8 kcal mol–1. However, the resulting intermediate **AI4** still lies lower in energy than **I2** by 4.5 kcal mol–1. In **AI4** the initial –N4H group in **I2** has

simple cleavage of the C—N bond. This occurs with a barrier of 7.3 kcal mol–1 relative to **AI4**  (**Table 7**). It should be noted that the resulting product complex of **AP** lies 11.8 kcal mol–1 lower in energy than **I2** and resembles a complex between NH3•+ and protonated

In contrast, in **Path B** the initial 'protonation' of N4 is achieved via an intramolecular rearrangement from the N3—H group and does not originate from solution. This reaction proceeds via the four-membered ring TS **BTS3** at a significant cost of 34.0 kcal mol–1 to give intermediate **BI3**, lying 8.8 kcal mol–1 higher in energy than **I2** (**Table 7**). However, similar to **Path A** the second step is protonation of N4 with the proton originating from solution. This process is exergonic with the resulting intermediate **BI4** lying 5.6 kcal mol–1 lower in free energy than **BI3**. The subsequent cleavage of the C4—N4H3+ bond occurs via **BTS4** to give the product complex **BP**. However, unlike for **Path A**, **BP** is higher in energy than **I2** by 7.1 kcal mol–1 and instead resembles a complex between NH3 and the oxidized enol form of Uracil. Like **Path A**, the alternate **Path C** begins with the exothermic protonation of N4 by a proton originating from solution to give **AI3** (**Table 7**). Now, however, the second proton is obtained via the intramolecular proton transfer that initiated **Path B**, i.e., by transfer from the N3—H group. This reaction proceeds via **CTS3** with a considerable barrier of 39.4 kcal mol–1, even higher than that observed in **Path B**, to give intermediate **BI4**. The remaining step, cleavage of the C4—N4 bond, is then identical to that of **Path B** as is the product

**Path D**, unlike the previous paths, involves an initial intramolecular proton transfer from the C4—OH hydroxyl onto the N4 centre (**Table 7**). This reaction proceeds via **DTS3** at a cost of 26.8 kcal mol–1. While this barrier is still quite high, it is 7.2 and 12.6 kcal mol–1 lower than the analogous intramolecular rearrangements in **Paths B** and **C** and is thus kinetically favoured. The resulting intermediate formed **DI3** lies only 2.2 kcal mol–1 higher in energy than **I2**. This is then followed by protonation of the N4 centre by a proton originating from solution, similar to **Paths A** and **B**. This step is exothermic with the intermediate formed, **DI4**, lying 6.4 kcal mol–1 lower in energy than **DI3**. The next and final step is the loss of NH3 and occurs via **DTS4** with a quite small barrier of just 2.1 kcal mol–1. The resulting product **DP**, resembling a complex between NH3 and oxidized uracil, is markedly lower in energy than **I2** by 26.3 kcal mol–1. Importantly, it is in fact the lowest energy product complex

The final path considered, **Path E**, begins with the same exothermic intermolecular protonation of N4 as in **Paths A** and **C** and again leads to formation of **AI3**. The next step, however, is intramolecular proton transfer from the C4—OH group to N4 as occurs in **Path D**. Importantly, it proceeds via **ETS3** with a higher barrier (33.9 kcal mol–1) than observed in **Path D** (26.8 kcal mol–1) to give **DI4**. The final step is then cleavage of the C4—N4 bond as in

Thus, while deamination via donation of both protons from the aqueous solution (**Path A**) may have the lowest associated barriers, the resulting product is not the most

+ group. Consequently, the next step is the loss of NH3 by

now formally become an –N4H3

obtained of all those considered herein.

**Path D** to give **DP**.

uracil.(Labet et al. 2008b)

formed.

thermodynamically favoured. In contrast, in those cases in which only one proton originates from solution (**Paths B – E**), only those involving intramolecular rearrangement of a proton from C4—OH (**Paths D** and **E**) are exothermic and lead to formation of the most thermodynamically preferred product (**DP**). Furthermore, the barrier for this rearrangement is lowest when it occurs prior to proton donation from the solvent. Lastly in the case where both protonations occur via intermolecular processes, NH3•+ is formed (**AP**) while NH3 is formed when only a single proton originates from exogenous sources (**BP** and **DP**).
