Glycosidic Bond Cleavage in DNA Nucleosides - ACS Publications

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Glycosidic Bond Cleavage in DNA Nucleosides: Effect of Nucleobase Damage and Activation on the Mechanism and Barrier Stefan A.P. Lenz, Jennifer Lee Kellie, and Stacey D Wetmore J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.5b10337 • Publication Date (Web): 30 Nov 2015 Downloaded from http://pubs.acs.org on December 1, 2015

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The Journal of Physical Chemistry

Glycosidic Bond Cleavage in DNA Nucleosides: Effect of Nucleobase Damage and Activation on the Mechanism and Barrier Stefan A. P. Lenz, Jennifer L. Kellie, and Stacey D. Wetmore* Department of Chemistry and Biochemistry, University of Lethbridge, 4401 University Drive West, Lethbridge, Alberta, Canada, T1K 3M4 Abbreviations: A, adenine; AAG, alkyladenine DNA glycosylase; BER, base excision repair; C, cytosine; EA, 1,N6-ethanoadenine; FPG, formamidopyrimidine DNA glycosylase; G, guanine; hNEIL1, human endonuclease VIII-like 1; hOgg1, human 8-oxoguanine DNA glycosylase; hUNG2, human uracil DNA glycosylase; Hx, hypoxanthine; MBD4, methyl-CpG binding domain protein 4; MutY, adenine DNA glycosylase; NTH1, endonuclease III-like 1; OG, 8-oxoguanine; potential energy surface, PES; RC, reactant complex; SI, supporting information; TDG, thymine DNA glycosylase; TS, transition state; U, uracil; UDG, uracil DNA glycosylase; X, xanthine; εA, 1,N6-ethenoadenine; εC, 3,N4-ethenocytosine; 3MeA, 3methyladenine; 7DzA, N7-deaza-adenine; 7DzG, N7-deaza-guanine;

AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. Tel.: (403) 329-2323. Fax: (403) 329-2057. Notes The authors declare no competing financial interests.

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ABSTRACT: Although DNA damage can have a variety of deleterious effects on cells (e.g., senescence, death, and rapid growth), the base excision repair (BER) pathway combats the effects by removing several types of damaged DNA. Since the first BER step involves cleavage of the bond between the damaged nucleobase and the DNA sugar–phosphate backbone, we have used density functional theory to compare the intrinsic stability of the glycosidic bond in a number of common DNA oxidation, deamination, and alkylation products to the corresponding natural nucleosides. Our calculations predict that the dissociative (SN1) and associative (SN2) pathways are nearly isoenergetic, with the dissociative pathway only slightly favoured on the Gibbs reaction surface for all canonical and damaged nucleosides, which suggests that DNA damage does not affect the inherently most favourable deglycosylation pathway. More importantly, with the exception of thymine glycol, all DNA lesions exhibit reduced glycosidic bond stability relative to the undamaged nucleosides. Furthermore, the trend in the magnitude of the deglycosylation barrier reduction directly correlates with the relative nucleobase acidity (at N9 for purines or N1 for pyrimidines), which thereby provides a computationally efficient, qualitative measure of the glycosidic bond stability in DNA damage. The effect of nucleobase activation (protonation) at different sites predicts that the positions leading to the largest reductions in the deglycosylation barrier are typically used by DNA glycosylases to facilitate base excision. Finally, deaza purine derivatives are found to have greater glycosidic bond stability than the canonical counterparts, which suggests that alterations to excision rates measured using these derivatives to probe DNA glycosylase function must be interpreted in reference to the inherent differences in the nucleoside reactivity. Combined with previous studies of the deglycosylation of DNA nucleosides, the current study provides a greater fundamental understanding about the reactivity of the glycosidic bond in damaged DNA, which has direct implications to the function of critical DNA repair enzymes.


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INTRODUCTION Despite nucleotides being the building blocks of DNA and RNA, as well as precursors for the synthesis of enzyme cofactors, chemical agents can damage these critical cellular components. In DNA, the nucleobases are particularly susceptible to damage, which can alter their base-pairing preferences. The consequences of nucleobase damage are varied, with mutations commonly resulting from the replication or transcription of damaged DNA. DNA damage that only slightly alters the chemical structure of a nucleobase (e.g. deamination, oxidation or alkylation) is typically repaired by the base excision repair (BER) pathway, which utilizes a host of enzymes to remove the damaged nucleotide and incorporate the correct nucleotide into the same position in the DNA strand.1-3 DNA glycosylases use a nucleophile to initiate this pathway by cleaving the glycosidic bond that connects the damaged nucleobase to the sugar–phosphate backbone.2,

4-5

This generates a cytotoxic abasic site, which is

subsequently processed by other BER enzymes. Due to the diversity in the structure and properties of damaged DNA nucleotides, there are many different DNA glycosylases in both eukaryotes and prokaryotes.2, 4-5 Each enzyme targets a specific subset of damaged nucleotides using unique active site–DNA interactions to facilitate substrate recognition and nucleobase removal. Since DNA is inherently stable, even following many types of damage, an abundance of experimental work has focused on the base removal BER step catalyzed by DNA glycosylases to better understand how these enzymes reduce the otherwise prohibitively high deglycosylation barrier.6-19 These works reveal that there are some common features in the function of this group of enzymes. Specifically, each enzyme relies on key active-site residues to lower the deglycosylation barrier through activation of the nucleophile,7, 10-11 stabilization of the positive charge that develops on the sugar,9, 12, 17 and activation of the nucleobase for departure (for example, through protonation,12,

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hydrogen bonding,15,

18

or non-specific π–π (stacking or T-shaped) interactions).18-19

However, the relative importance of the proposed factors employed to reduce the deglycosylation


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barrier depends on the glycosylase considered. For example, UDG is proposed to facilitate removal of uracil by using hydrogen bonds to stabilize the oxocarbenium transition state and the departing (negatively charged) nucleobase,9,

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MutY has been proposed to directly protonate the departing

adenine nucleobase,12, 21 while AAG has been proposed to use active site π–π contacts to identify and remove a wide variety of substrates.18, 22 It is not clear whether these differences are connected to the properties of the nucleophile, nucleobase and/or active site residues. To complement experimental work on BER, large-scale computer modeling has provided insights into the deglycosylation mechanism employed by several DNA glycosylases, including MutY,21, 23 AAG,22, 24

hOgg1,25-26 FPG,27-28 TDG,29 and hUNG2.30 Although these works provide useful insight into the activity

of specific enzymes towards a particular substrate, it is difficult to extrapolate general information about the individual contribution of various factors to the barrier reduction necessary to promote base excision. Therefore, our group has studied the fundamentals of the glycosidic bond cleavage in DNA using minimal models that do not reflect a specific glycosylase.31-38 Specifically, we have examined the effects of the nucleophile by considering different hydrolysis mechanisms,31, nucleophile (water) activation,31-32,

35-37

33-34

different levels of

and different nucleophiles (water versus an amine-containing

residue).35, 39-40 The effect of the nucleobase has been evaluated by considering deglycosylation of each canonical DNA nucleoside,31-32 as well as different (anti/syn) relative orientations of the nucleobase about the glycosidic bond.35 Furthermore, the magnitude of the effect of different levels of nucleobase activation on the barrier was determined by hydrogen-bonding small molecules (hydrogen fluoride, ammonia or water) to different nucleobase sites,31-32, 36 as well as stacking model amino acid side chains (benzene) with respect to the nucleobase.36 The effects of other environmental contributions, including solvation32-37 and the DNA backbone,37 have also been investigated. Despite these studies providing critical information about the intrinsic effects of the nucleophile, nucleobase activation and the environment on the structure and energetics of stationary points along the DNA deglycosylation


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pathway, the effect of DNA damage on the reaction barrier and mechanism remains unclear. Indeed, to the best of our knowledge, while the deglycosylation pathway has been considered for select DNA nucleobases that are excised by DNA glycosylases, including uracil,31-34,

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8-oxoguanine,35,

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8-

oxoadenine,42 and oxidized thymine,38, 43-44 no study to date has compared the deglycosylation of a semicomprehensive list of damaged DNA nucleotides to their corresponding natural (undamaged) counterparts. In the present work, we continue to map the essential components of the DNA deglycosylation reaction by comparing the intrinsic reactivity of the glycosidic bond in established substrates for wellstudied DNA glycosylases to the canonical nucleosides (Figure 1). The damaged derivatives considered include some of the most mutagenic lesions arising from DNA oxidation, deamination, and alkylation. Specifically, we investigate the stability of damage repaired by AAG,7, 45 including 1,N6-ethenoadenine (εA), 1,N6-ethanoadenine (EA), 3-methyladenine (3MeA), xanthine (X), 7-methylguanine (7MeG), and hypoxanthine (Hx). Furthermore, we examine common DNA damage products, including 1) 8oxoguanine (OG), which is repaired by glycosylases such as hNEIL1, hOgg1 and Fpg;4-5 2) uracil (U), which is excised by (bacterial) UDG and (human) hUNG2;46-48 3) εC, which is removed from DNA by TDG;49-50 and 4) (5R,6S)-thymine glycol (Tg), which is repaired by hNEIL1, TDG, and MBD4.4-5,

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Finally, we

consider the purine derivatives formed by replacing N7 with a carbon atom, namely 7-deaza-A (7DzA) and 7-deaza-G (7DzG), since these modified nucleobases are commonly used to monitor enzymatic function.45, 52-54 Using computational models previous established to provide critical information about deglycosylation in DNA nucleosides,31-32,

35-37

we study both concerted and dissociative pathways to

compare these two potential deglycosylation mechanisms. The effects of nucleobase protonation (activation) are also considered at different nucleobase sites to determine whether the most beneficial activation points correlate with the sites proposed to be critical for DNA glycosylase function. Together, this work provides important insights into the inherently preferred deglycosylation pathway and


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energetics for damaged DNA nucleosides. When combined with experimental and computational studies on a wide range of DNA glycosylases, this study will enhance our understanding of the role of the glycosidic bond stability in the relative excision rates for different substrates and the greater persistence of some lesions in the genome. Furthermore, our work serves as a useful reference point for future large-scale modeling of a variety of DNA glycosylases involved in a key cellular repair process in humans.

COMPUTATIONAL DETAILS In the present study, we focus on the hydrolysis of the glycosidic bond in DNA nucleosides since many (monofunctional) DNA glycosylases employ a water nucleophile to facilitate base excision.2, 4-5, 55 Our corresponding model was carefully designed based on the most efficient computational model previously established for DNA deglycosylation (Figure 2).31-32, 37 Specifically, we used formate-activated water as the nucleophile since a measure of water activation has been determined to be required to accurately model hydrolytic deglycosylation. A formate residue was chosen for our model since a conserved Asp or Glu typically functions as the general base within the active site of (monofunctional) DNA glycosylases, and has also been proposed to facilitate the reaction through stabilization of the positive charge developing on the deoxyribose moiety during the reaction.56-58 d nucleosides repaired by glycosylases that do not employ a Asp or Glu as the general base, namely bifunctional hOgg1, hNEIL1, and Fpg, as well as monofunctional TDG. Nevertheless, the active sites of hOgg1, hNEIL1, and Fpg contain an active site Asp or Glu that serves to stabilize the cationic charge developing on the sugar during the deglycosylation reaction. Therefore, our model provides valuable information about the intrinsic stability of the glycosidic bond in each system investigated. The model deoxyribose moiety is substituted at the O3′ and O5′ positions with methyl groups since capping with hydrogen atoms has


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been shown to lead to interactions with the nucleobase that cannot occur in the DNA helix,31 and inclusion of the phosphate moieties has been determined to be an unnecessary computational cost.37 Initial guesses for the methyl-capped canonical nucleosides were obtained from our previous work,37 with the nucleobase modified to form the damaged derivatives considered in the present contribution (Figure 1). Each structure was optimized using the B3LYP functional and the 6-31+G(d) basis set in water using the IEF-PCM solvation model (ε = 78.36). Gaussian 09 defaults for the convergence criteria were implemented (i.e., the maximum force must be < 0.000450 au, the RMS force < 0.000300 au, the maximum step size < 0.001800 au, and the RMS step size < 0.001200 au). This approach was chosen due to our previous successes using the same level of theory to study canonical nucleoside deglycosylation,31-38 and evidence that the inclusion of implicit solvent during the optimization step affects the structure of stationary points along the hydrolysis pathway.37 Relative energies were obtained from scaled (0.9806) zero-point energy corrected IEF-PCM-B3LYP/6-311+G(2df,2p) single-point calculations, and relative Gibbs energies were obtained from thermally-corrected SMD-B3LYP/6311+G(2df,2p) single-point calculations. Although most previous computational studies of DNA deglycosylation have focused on the concerted pathway,32,

37, 59-60

experimental evidence suggests that non-enzymatic hydrolysis likely proceeds

through a dissociative mechanism for undamaged DNA.61-64 Therefore, both the concerted (SN2) and stepwise (SN1) pathways are characterized in the present work to determine the favoured deglycosylation mechanism (Figures 3–5 and S1–S6). Transition states (TS) corresponding to the concerted pathway were fully optimized, and intrinsic reaction coordinate (IRC) calculations were used to confirm that the isolated structures are directly connected to the reported reactant complexes (RC), and product complexes (PC, Figures S7–S9). For each deglycosylation reaction, the product complexes verify that complete nucleobase dissociation, and nucleophile association has occurred in conjunction with proton transfer from the nucleophile to the formate anion. Previous work has determined that 1D


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Gibbs energy scans generated using the glycosidic bond as the reaction coordinate are sufficient for modelling SN1 nucleoside deglycosylation.33 Therefore, the SN1 transition states were identified by holding the glycosidic bond fixed in 0.200 Å increments (from 1.300 Å to 3.100 Å) starting from the reactant complexes identified from the IRC calculations for the SN2 transition states. Once an energy maximum was identified on the Gibbs reaction surface, frequency calculations were performed to ensure that the structure corresponds to an SN1 transition state. During these scans, the nucleophile was unconstrained and thereby allowed to adopt the position that provides maximum charge stabilization to the oxocarbenium transition state. Nevertheless, in each transition state, the nucleophile is far removed from the sugar moiety and therefore we are confident the reported structures correspond to dissociative pathways. We note that our reported intermediate structures (Figures S7–S9) are estimates from very flat reaction surfaces obtained by further lengthening the glycosidic bond. These estimated intermediates typically differ from the corresponding transition states by < 5 kJ mol-1 and the glycosidic bond distances differ by 0.200 – 0.600 Å. However, these results are in close agreement with our previous work that modelled non-enzymatic33-34, 38 or enzymatic deglycosylation22, 30 reactions using 2D reaction surfaces. Specifically, for a range of nucleosides, we previously determined that the dissociative transition state and corresponding intermediate typically differ by only 0.400 Å, while the dissociative intermediate was either unable to be identified,30 or these structures were only slightly more stable than the corresponding transition state.22,

33-34, 38

Therefore, we are confident that the reported

structures are accurate estimates of the transient dissociative intermediates. Most importantly, these intermediates verify that the reported transition structures correspond to nucleobase departure. All calculations were carried out using Gaussian 09 (Revision A.02 or D.01).65

RESULTS AND DISCUSSION


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Effect of Nucleobase Damage Adenine Derivatives. The calculated structures of the RC and TS for the (concerted and dissociative) hydrolysis of adenine, common types of adenine damage, and the 7DzA analogue are shown in Figure 3. The adenine reactant complex (A-RC, Figure 3) has a glycosidic bond length of 1.458 Å, a nucleophile– sugar (C1′) distance of 3.981 Å, and a reaction angle (∠(OC1′N9)) of 145.1°. The water nucleophile hydrogen bonds to O3′ of the sugar and the formate anion, and resides on the opposite side of the sugar as the nucleobase and O5′, while the formate anion forms a hydrogen bond with H4′. The overall structure of the reactant complex is consistent for each type of adenine damage considered. The reactant complex corresponding to the neutral damaged adenine products (Hx, εA and EA) are very similar to that of canonical A, with glycosidic bond lengths of ∼ 1.46 Å, nucleophile–sugar (C1′) distances of ∼ 4.0 – 4.1 Å, and reaction angles of ∼ 146 – 148°. Although the glycosidic bond length in the RC for cationic 3MeA is only 0.019 Å longer than for canonical A, the nucleophile–sugar distance is significantly (0.616 Å) shorter, which likely reflects stabilization of the cationic methyl-capped nucleoside by the anionic (formate–water) nucleophile. In contrast, both the glycosidic bond and nucleophile–sugar distances are shorter in the 7DzA reactant complex than for canonical A (by 0.011 and 0.280 Å, respectively), which hints that this nucleoside may exhibit a different susceptibility to deglycosylation. The SN2 transition state associated with A (Figure 3) contains a glycosidic bond distance of 2.594 Å, a nucleophile–sugar distance of 2.538 Å, and a reaction angle of 149.8°. Despite the smaller nucleophilic distance, water adopts a similar orientation in the TS as the RC and is in a good position for nucleophilic attack. However, since the nucleophile complex is closer to the sugar moiety in the concerted TS than the RC, neither the formate anion nor water hydrogen bonds with the sugar moiety (Figure 3). Furthermore, the nucleophilic water retains the proton in the transition state, although the hydrogen bond between water and the formate ion shortens (by approximately 0.05 Å) and the water


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O–H bond elongates by 0.01 Å compared to the reactant complex. The corresponding TS for both Hx and EA have similar reaction parameters to the A–SN2 TS, with a less than 0.1 Å difference in the glycosidic bond and nucleophilic distances, and a less than 0.7° change in the reaction angle (Figure 3). On the other hand, the transition states for εA and 3MeA occur slightly earlier along the deglycosylation pathway, with glycosidic bond distances of ∼ 2.3 Å, nucleophilic distances of ∼ 2.8 Å, and reaction angles of ∼ 150 – 157°. Compared to canonical A, the SN2 transition state is later for 7DzA, with ∼ 0.2 Å longer glycosidic bond and shorter nucleophile–sugar distances. On the SN1 Gibbs surface for the hydrolysis of a canonical or neutral damaged adenine nucleoside (Figure S1B, Supporting Information (SI)), the energy begins to plateau at approximately 2.500 Å and the energy reaches a maximum at approximately 2.700–2.900 Å. The energy maximum occurs earlier (2.500 Å) for 3MeA (Figure S1A, SI) compared to neutral canonical or damaged A, likely owing to the greater charge stabilization of the (initially) cationic nucleobase upon deglycosylation. In each SN1 transition structure (Figure 3), the nucleophilic distance is ∼ 4.2 Å. As discussed for the reactant complexes, the formate-activated water remains on the opposite side of the sugar as the nucleobase, providing charge stabilization to the oxacarbenium cation forming during deglycosylation, but does not associate with C1′. Therefore, these structures indeed correspond to dissociative transition states. The ∠(OC1′N9) reaction angle also remains consistent across the adenine derivatives, adopting a value of 167 – 169° for all nucleobases except 3MeA (150.9°). With the exception of Hx and 3MeA, there is a significant rotation of the nucleobase with respect to the sugar moiety along the dissociative transition state upon glycosidic bond cleavage, which typically maximizes stabilizing hydrogen bonds between the sugar and the departing nucleobase (Figure 3). According to our model, the barrier for A deglycosylation is 143.7 kJ mol–1 for the concerted pathway and 148.1 kJ mol–1 for the dissociative pathway (∆‡E, Table 1). When A is damaged, the


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hydrolysis barrier decreases regardless of the mechanism. The barrier reduction is largest (~ 65 kJ mol–1; ∆mod, Table 1) for (cationic) 3MeA, which is unsurprising since the cationic lesion can better accommodate the negative charge accumulating on the nucleobase during deglycosylation. However, both Hx and εA also have an up to 11 kJ mol–1 smaller deglycosylation barrier compared to undamaged adenine for both pathways. In contrast, the deglycosylation barrier for EA is within 1 kJ mol–1 of that for canonical A, which further highlights the different reactivity exhibited by various DNA damage products. When the Gibbs reaction energetics are considered (∆‡G, Table 1), the concerted barrier for deglycosylation of (undamaged) A decreases by 0.7 kJ mol–1, while the dissociative barrier decreases by 15.3 kJ mol–1, compared to the PES. The greater stabilization exhibited for the SN1 pathway occurs in part since precise energy maxima are found earlier on the Gibbs energy surfaces (Figure S1A, SI) compared to the later plateaus on the PES (Figure S1B, SI). The earlier transition states on the Gibbs energy surfaces are a consequence of the inclusion of short-range interactions between the solvent and solute in the SMD approach,66 which provides enhanced stabilization for charge-separated species and therefore greater stability later in the deglycosylation pathway. When the damaged A derivatives are considered, the ∆‡G barriers are up to 10 kJ mol–1 less than the corresponding ∆‡E values for the concerted pathway, and up to 24 kJ mol–1 less than the corresponding values for the dissociative pathway (Table 1). Regardless, the trends and approximate magnitude of the effects of nucleobase damage on the A deglycosylation barrier are the same on the Gibbs and potential energy surfaces, falling between the extremes of EA reducing the barrier by less than ~ 8 kJ mol–1 and 3MeA decreasing the energetic requirement by up to ~ 67 kJ mol–1 (∆mod, Table 1). Interestingly, the calculated trends in the deglycosylation barriers as a function of adenine damage correlate with the calculated N9 acidities (∆+, Table 1). Specifically, the deglycosylation barrier on the Gibbs energy surface decreases as A ≈ EA > εA ≈ Hx > 3MeA, while the acidity increases as A ≈ EA


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< εA ≈ Hx < 3MeA. Furthermore, the changes in the barrier and acidity upon damage are of the same order of magnitude. For example, Hx is –19.6 kJ mol–1 more acidic than A and the corresponding damaged hydrolysis barrier is –15.4 to –16.5 kJ mol–1 lower than the A barrier depending on the pathway. In contrast, 3MeA is –56.4 kJ mol–1 more acidic than A and the corresponding deglycosylation barrier is –64.5 to –67.4 kJ mol–1 smaller depending on the mechanism (Table 1). Therefore, although there is not perfect agreement between the magnitude of the effect of the nucleobase damage on the acidity and deglycosylation barrier, the calculated acidities provide a computationally efficient and meaningful qualitative estimate of the deglycosylation barrier relative to undamaged A. In terms of the preferred deglycosylation pathway, the SN2 mechanism is slightly favoured over the SN1 pathway (by ∼ 1 – 4 kJ mol–1; ∆mech, Table 1) on the potential energy surface. However, this trend reverses on the Gibbs energy surface such that the dissociative pathway is favoured over the concerted mechanism (by ∼ 7 – 24 kJ mol–1; ∆mech, Table 1). This latter result is consistent with experimental data for the non-enzymatic deglycosylation of nucleic acids, which suggests that the reaction likely proceeds via an SN1 mechanism.61, 64, 67 Our results also indicate that the inclusion of the entropic term (as calculated by the SMD solvation approach) greatly contributes to the deglycosylation barrier, although we acknowledge that SMD solvation can over-estimate the entropic contribution.68 In contrast to the non-enzymatic reaction, AAG (the glycosylase responsible for catalyzing the excision of εA, EA 3MeA, and Hx) has been shown to facilitate removal of εA and 3MeA through an SN2 mechanism22 (pathway for Hx and EA removal has yet to be determined). Despite glycosidic bond hydrolysis in these modified nucleosides occurring through a dissociative mechanism, AAG may facilitate base excision in part by using nucleobase–active site interactions to stabilize an intrinsically less favourable (non-enzymatic) mechanism. Nevertheless, mechanistic studies of MutY-mediated excision of adenine reveal that the reaction proceeds via an SN1 reaction,6 which highlights that at least some glycosylases employ the inherently more stable deglycosylation mechanism to fulfill their function.


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Although not a form of damaged DNA, deglycosylation within the 7DzA nucleoside is of interest due to the common use of this modification to study the binding and function of DNA glycosylases.45, 52, 54

As discussed above, the structures along the deglycosylation pathway corresponding to 7DzA are

varied compared to those for canonical or damaged A. Furthermore, Table 1 indicates that 7DzA is the lone exception to the deglycosylation trend, with the modification increasing the barrier compared to A deglycosylation by 26.2 – 27.4 kJ mol–1 on the PES and 18.1 – 22.6 kJ mol–1 on the Gibbs energy surface. The larger barriers for 7DzA compared to A are consistent with the replacement of an electron-rich nitrogen atom with a carbon atom, rendering the nucleobase a poorer leaving group due to the inability to delocalize charges forming during deglycosylation. Therefore, although endocyclic nitrogen atoms are often replaced with carbon atoms as a tool to identify nucleobase sites that are activated during enzyme-catalyzed deglycosylation, changes in the intrinsic reactivity of the glycosidic bond mean that observed rate reductions may be misleading. Caution must be exercised when using these modified nucleobases to evaluate the chemistry catalyzed by DNA glycosylases.

Guanine Derivatives. The reactant complexes for canonical and damaged guanine deglycosylation (Figure 4) resemble those discussed for the adenine derivatives (Figure 3). Notably, the glycosidic bond distances range from 1.45 to 1.48 Å, while the nucleophile resides in a similar position with respect to the sugar moiety ( ∼ 4.0 – 4.1 Å from C1′ on the opposite side as the nucleobase), and hydrogen bonds with O3′ via water and H4′ via the formate anion. Furthermore, there is slightly greater variation in the reaction angle along the G series (∼ 136 – 149°). Unlike discussed for the A derivatives, 7MeG and 7DzG also fit within this general structural trend for the RC. The TS for the G and A derivatives are also very similar (Figures 3 and 4). Specifically, the SN2 transition states for neutral OG has a glycosidic bond length of 2.678 Å, and a nucleophilic distance of ∼ 2.482 Å, which breaks key hydrogen bonds with the


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sugar moiety observed in the RC. In the OG SN1 transition state, the formate-activated water nucleophile maintains the hydrogen bonding with H4′ and O3′ observed in the reactant complex, and is located ∼ 4.231 Å from C1′, while the glycosidic bond length is 3.100 Å, as seen for the A derivatives. However, unlike 7DzA, the structure of the 7DzG transition states fall within the general trends for the neutral G and OG derivatives. Furthermore, although the nucleophile adopts a similar orientation relative to the nucleoside, the transition state occurs earlier for X compared to OG along both the concerted (glycosidic bond and nucleophilic distances of 2.533 Å and 2.557 Å, respectively) and dissociative (glycosidic bond and nucleophilic distances of 2.900 Å and 4.275 Å, respectively) mechanisms, which highlights the greater reactivity of the associated glycosidic bond in this neutral lesion. Nevertheless, as discussed for 3MeA, 7MeG has the earliest TS in both the concerted (glycosidic bond and nucleophilic distances of 2.406 Å and 2.735 Å, respectively) and dissociative (glycosidic bond and nucleophilic distances of 2.500 Å and 4.921 Å, respectively) deglycosylation pathways, due to the ability of the cationic nucleobase to accommodate the negative charge developing during the hydrolysis reaction. Our model predicts the barrier for G excision on the PES to be 145.8 kJ mol–1 for the concerted mechanism and 148.3 kJ mol–1 for the dissociative pathway (∆‡E, Table 1). The corresponding concerted and dissociative barriers on the Gibbs energy surface are 146.0 and 129.1 kJ mol–1 (∆‡G, Table 1), with the significant change in the dissociative barrier arising due to the earlier TS on the Gibbs surface compared to the PES (Figure S2, SI). These barriers differ from those for A by less than 3 kJ mol–1, which reflects the similarity in the nucleobase (N9) acidity of the canonical purines (differ by 1.2 kJ mol–1; Table 1). As observed for A, damage to G reduces the barrier for excision regardless of the hydrolysis mechanism (∆mod, Table 1), with the magnitude of the barrier reduction most significantly dependent on the nucleobase modification. Specifically, although OG is the most stable form of G damage considered in the present work, the corresponding ∆‡G deglycosylation barrier is ∼ 10 kJ mol–1 smaller for the concerted pathway, and ∼ 17 kJ mol–1 smaller for the dissociative mechanism, than predicted for G.


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However, the barrier reduction is even more significant for the X lesion, being up to ∼ 23 – 26 kJ mol–1 regardless of the reaction pathway, which is in agreement with our calculated structures, as well as the instability of X previously measured in solution,69 and the shorter half-life (relative to G) of the X glycosidic bond in the DNA helix.64,

70

Indeed, the intrinsic instability of glycosidic bond in the X

nucleoside is also reflected in the calculated acidity (30.8 kJ mol–1 more acidic than G, Table 1). Unsurprisingly, the deglycosylation barrier reduction is greatest for cationic 7MeG (∼ 53 – 57 kJ mol–1). Overall, the trend in barrier reductions (7MeG > X > OG) correlates with the effect of the damage on the N9 acidity of G (Table 1), and supports the use of this property as a measure of inherent differences in the reactivity of the glycosidic bonds in natural and damaged DNA. In terms of the preferred deglycosylation mechanism, the G deglycosylation barrier is lower for the concerted pathway on the PES (by 2.5 kJ mol–1), but lower for the dissociative pathway on the Gibbs surface (by 16.9 kJ mol–1; ∆mech, Table 1). This trend is consistent regardless of the type of G damage considered, with the SN2 pathway being favoured by 0.2 – 3.8 kJ mol–1 on the PES and the SN1 pathway being preferred by 13.7 – 23.8 kJ mol–1 on the Gibbs surface. These results contrast those for A derivatives, where only a slight (up to 7 kJ mol–1) shift toward the dissociative pathway was observed on the Gibbs surface. Therefore, although there is a consistent preference for a dissociative mechanism on the Gibbs energy surfaces among the canonical and damaged A and G nucleosides, the SN1 pathway is overall more favoured for damaged G lesions than for damaged A derivatives. Despite this intrinsic preference for an SN1 mechanism, both AAG and hOgg1 have been proposed to catalyze SN2-mediated excision of damaged purine derivatives.18, 22, 71-73 As discussed for 7DzA, 7DzG is of interest due to the use of such modifications to garner information about the function of DNA glycosylases.45, 52-54 Unlike discussed for canonical or damaged G, the 7DzG lesion exhibits a deglycosylation barrier of ∼ 170 – 174 kJ mol–1 on the PES and ∼ 156 – 170 kJ


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mol–1 on the Gibbs energy surface. Therefore, this G derivative has the most stable glycosidic bond among those considered in the present work, being ∼ 23 – 27 kJ mol–1 more stable than the canonical nucleoside. These results further support our proposal that replacement of an endocyclic nitrogen atom with a carbon atom can dramatically change the intrinsic chemistry of the nucleoside, resulting in significantly higher reaction barriers. Therefore, changes in DNA glycosylase activity due to such modifications must be interpreted with this effect in mind rather than directly correlating changes in the activity to the role of discrete active site interactions.

Pyrimidine Derivatives. The general structural features of the RC and TS for the deglycosylation in the pyrimidine derivatives (Figure 5) mirror those discussed for the purine derivatives (Figures 3 and 4). Specifically, regardless of whether natural or damaged C or T are considered, the reactant complex exhibits a glycosidic bond length of ∼ 1.46 – 1.48 Å, a nucleophile distance of ∼ 3.9 – 4.3 Å, and a reaction angle of 143 – 153°. The formate–activated water is situated on the opposite side of the deoxyribose moiety as the nucleobase, and held in place through hydrogen bonds between H4′ and the formate ion, and O3′ and water. The nucleophile complex moves closer to C1′ in all concerted TS for the pyrimidine derivatives, which breaks hydrogen-bonding interactions with the sugar moiety. However, the damaged species have later SN2 transition states than undamaged cytosine or thymine (Figure 5). Specifically, hydrolysis in the natural nucleosides occurs with a glycosidic bond length of ∼ 2.4 – 2.5 Å, and a nucleophile distance of ∼ 2.6 – 2.7 Å. In contrast, the glycosidic bond length is ∼ 2.7 Å and the nucleophile distance is ∼ 2.5 Å in the concerted TS of the damaged nucleosides. Nevertheless, regardless of whether the natural or damaged pyrimidine derivatives are considered, the dissociative TS adopts a consistent structure with a glycosidic bond length of 2.900 Å, nucleophile distance of ∼ 4.2 – 4.3 Å, and


16

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the nucleophile is positioned relative to the nucleoside in a manner consistent with the corresponding RC (Figures 5 and S3). The ∆‡G (∆‡E) deglycosylation barrier for canonical C is 143.4 (150.5) kJ mol-1 for the concerted pathway and 126.2 (151.2) kJ mol-1 for the dissociative pathway (Table 1). These barriers are larger (by up to ∼ 24 kJ mol–1) than those for undamaged T, which are 119.3 (131.4) kJ mol-1 for the SN2 and 112.6 (138.3) kJ mol-1 for SN1 mechanism. This trend correlates with the calculated (N1) acidity being 29.0 kJ mol–1 greater for T than C (Table 1). As discussed for the purine derivatives, the damaged C lesions have smaller hydrolysis barriers than the natural C counterpart. Specifically, the barrier reductions are comparable for both forms of C damage, being ∼ 20 – 22 kJ mol–1 on the PES and ∼ 22 – 27 kJ mol–1 on the Gibbs energy surface (∆mod, Table 1), which arises since U and εC are significantly more acidic than C (by ∼ 27 – 33 kJ mol–1), but are similar in acidity to each other (∆mod, Table 1). In contrast, the deglycosylation barrier associated with Tg is up to ∼ 24 kJ mol–1 greater than the corresponding barrier for T, which makes Tg the only naturally-occurring damaged nucleobase considered in the present work that is more stable than its corresponding undamaged counterpart. Nevertheless, this finding is consistent with the (27.0 kJ mol–1) greater calculated acidity for Tg than T (Table 1), and previous work comparing the deglycosylation barrier of T and Tg in water.38 This unique result suggests DNA glycosylases that target Tg (e.g. hNTH1, hNEIL1, TDG and MBD4)74-75 must overcome an otherwise significant barrier to deglycosylation in order to facilitate base excision of damaged DNA. Interestingly, each of the glycosylases that target Tg listed above have additional substrates, and therefore, these enzymes must balance their efficiency towards Tg and their specificity for other substrates.4-5, 76 In terms of the preferred deglycosylation pathway (∆mech, Table 1), the concerted and dissociative barriers are within 1 kJ mol–1 for C and its derivatives on the PES, while the SN1 mechanism is favoured by ∼ 17 – 19 kJ mol–1 on the Gibbs energy surface. On the other hand, T and Tg show a stronger


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preference (by ∼ 7 – 10 kJ mol–1) for the concerted pathway on the PES than the C derivatives, but a similar preference for the dissociative mechanism (up to ∼ 20 kJ mol–1) on the Gibbs energy surface. These results are consistent with those discussed above for the neutral purine derivatives. Therefore, regardless of the canonical nucleobase or damaged type considered, DNA nucleosides inherently prefer a dissociative pathway for deglycosylation facilitated by formate–activated water. This finding is largely consistent with the proposed mechanisms for DNA glycosylases that target pyrimidine derivatives. Specifically, U has been proposed to be removed by hUNG2 in humans or UDG in bacteria through an SN1 pathway,5,

30, 77-78

which correlates with our predicted preferred inherent chemistry (Table 1).

Furthermore, a dissociative mechanism has been shown to be favoured for TDG-mediated excision of mismatched thymine.29 Nevertheless, the significant barriers calculated in the absence of specific active site contacts for pyrimidine derivatives suggests that these repair enzymes must provide significant charge stabilization (beyond the Asp residue that activates the water nucleophile) to facilitate base excision.

Effect of Nucleobase Activation As discussed in the Introduction, key active site–nucleobase interactions have been implicated in the mechanism of action of several DNA glycosylases, with the primary assigned role being stabilization of the charge developing on the nucleobase during the deglycosylation reaction.12, 15, 18, 20 This stabilization may come from hydrogen bonding with the nucleobase15, 18 or full proton transfer to the nucleobase.12, 20 Furthermore, the proposed site of nucleobase activation varies between different enzymes.12, 20, 45 Due to the proposed importance of nucleobase stabilization along the deglycosylation reaction from these experimental studies, as well as previous computational works,31-32,

36-37

we

examined the effects of nucleobase activation (protonation) on the deglycosylation mechanism and


18

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barrier for the canonical and select damaged nucleosides (Table 2 and Figures S4 – S6, SI). It should be noted that protonation of the nucleobase represents the extreme of nucleobase activation, and that the barrier for nucleoside deglycosylation facilitated by multiple hydrogen bonds likely falls between the unassisted and fully protonated pathways.31-32, 36-37 In terms of the canonical purines, the effect of nucleobase activation (protonation) is very similar regardless of the nucleobase (A or G), mechanism (SN2 or SN1) or surface (potential or Gibbs energy) considered (∆+, Table 2). Specifically, nucleobase protonation decreases the barrier by up to ∼ 70 kJ mol–1, with activation at N7 leading to a larger barrier reduction (∼ 55 – 70 kJ mol–1) than N3 (∼ 40 – 60 kJ mol–1). In A, the N1 site is also available for protonation, which leads to the smallest barrier reduction (∼ 32 – 37 kJ mol–1). This trend in protonation directly correlates to the N9 acidities for the canonical purines, with the greatest acidity occurring upon N7 protonation (∆+, Table 2). Furthermore, this trend is maintained upon consideration of εA, where protonation at N7 leads to the largest (∼ 53 – 66 kJ mol–1) decrease in the hydrolysis barrier, as well as N9 acidity. In contrast, N7 protonation is not possible in OG (Figure 1). Instead, O8 protonation has a larger effect on the OG deglycosylation barrier (∼ 40 – 80 kJ mol–1 depending on whether the proton is oriented towards N7 or N9, as well as the mechanism and surface considered) than protonation at N3. In terms of the pyrimidines derivatives, O4 protonation can significantly reduce the deglycosylation barrier height associated with T and U (by ∼ 33 – 60 kJ mol–1). Nevertheless, O2 protonation is even more beneficial for T and U, as well as C, with each base exhibiting a barrier reduction of ∼ 61 – 88 kJ mol–1. Interestingly, despite the changes in the barrier height upon protonation, the preferred mechanism generally remains unaltered compared with the corresponding unactivated deglycosylation mechanism. Specifically, regardless of the nucleobase protonation state and site, the concerted pathway is favoured on the PES, while the dissociative pathway is favoured on the Gibbs energy surface (∆mech,


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Table 2). Therefore, even full protonation within the enzyme active site may not be sufficient to stabilize a different deglycosylation pathway. Most interestingly, the nucleobase sites that are most favourable for catalysis (i.e., afford the greatest barrier reductions following protonation) are generally those utilized by DNA glycosylases to facilitate DNA repair. For example, MutY targets the removal of A that has been mispaired opposite OG.79 The proposed mechanism of action of MutY points toward protonation of A at N7 in conjunction with base excision,6 which the present work indicates is the protonation site that leads to the largest inherent barrier reduction. Similarly, AAG has been proposed to facilitate removal of εA through a water chain that may protonate the damaged base at N7.45 In contrast, OG is removed by hOgg1 in humans and FPG in bacteria.80-81 Although no experimental work to date supports hOgg1 using O8 as a point of OG activation during the deglycosylation reaction,71, 73 FPG has been proposed to activate OG through hydrogen bonding at O8 with a backbone amide.82 Finally, the greater barrier reduction predicted by our model for protonation of U at O2 than O4 is consistent with experimental evidence suggests that hUNG2 in humans30, 48, 83 and UDG in E. coli.10, 84 use a histidine residue (His268 and His187, respectively) to stabilize negative charge developing on the nucleobase during deglycosylation through a strong hydrogen bond with O2. These select examples emphasize that the DNA glycosylases in many cases may exploit the inherent chemistry of damaged DNA to facilitate base excision repair.

CONCLUSIONS The current study examined the effect of DNA nucleobase damage on the inherent pathway and energetics for hydrolysis of the DNA glycosidic bond. We focused on nucleobase derivatives that are common DNA glycosylase substrates and span different (oxidation, deamination, and alkylation) damaged products. We determine that the dissociative and associative mechanisms are nearly


20

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isoenergetic, with the dissociative mechanism only slightly preferred on the Gibbs energy surface for all canonical and damaged nucleosides. Therefore, DNA damage does not change the inherent deglycosylation mechanism. Interestingly, comparison to previously proposed mechanisms for select glycosylases indicates that some enzymes take advantage of the intrinsically preferred pathway, while others will stabilize an inherently less favourable mechanism. Nevertheless, DNA damage lowers the deglycosylation barrier regardless of the pathway. This suggests that DNA glycosylases can exploit changes in the inherent chemistry of damaged DNA to facilitate base excision repair. The only exception is Tg, which has a larger hydrolysis barrier than T. The magnitude of the barrier reduction depends on the damaged nucleobase, and the trend in the barrier reduction correlates with differences in the (N9 for purines and N1 for pyrimidine) acidities, which provides a computationally efficient and meaningful qualitative estimate for the inherent barrier reduction due to damage formation. A correlation is revealed between the nucleobase activation (protonation) sites that lead to the largest barrier reductions and the sites invoked during enzymatic cleavage along the BER pathway for select canonical and damaged nucleobases. Finally, the stabilities of the glycosidic bond in deaza purine nucleosides (7DzA and 7DzG) are shown to be significantly different from the natural nucleosides, and therefore conclusions arising from analysis of DNA glycosylase function based on these derivatives must be carefully reconsidered. In summary, the present study provides key information about the intrinsic chemistry of damaged DNA that has implications for understanding DNA repair enzymes.

ACKNOWLEDGMENTS We thank the Natural Sciences and Engineering Research Council of Canada (NSERC, 249598-07), Canada Research Chain Program (950-228175), and Canada Foundation of Innovation (22770) for financial support. S.A.P.L. acknowledges NSERC (USRA), while J.L.K acknowledges NSERC (CGS-D) and the


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University of Lethbridge, for student scholarships. Computational resources from the Upscale and Robust Abacus for Chemistry in Lethbridge (URACIL) and those provided by Westgrid and Compute/Calcul Canada are greatly appreciated.

ASSOCIATED CONTENT Supporting Information Potential and Gibbs energy surfaces for the dissociative hydrolysis of canonical and damaged nucleosides (Figures S1 – S6); Full citation for reference 65. This material is available free of charge via the Internet at http://pubs.acs.org.

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65.

66.

67. 68. 69.

70. 71. 72. 73. 74.

75.

76.

77. 78.

79. 80. 81.

82. 83.

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Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G. E.; Robb, M. A.; Cheeseman, J. R.; Scalmani, G.; Barone, V.; Mennucci, B.; Petersson, G. A.; et al. Gaussian 09, Revision A.02; Gaussian, Inc.: Wallingford CT, 2009. Marenich, A. V.; Cramer, C. J.; Truhlar, D. G., Universal Solvation Model Based on Solute Electron Density and on a Continuum Model of the Solvent Defined by the Bulk Dielectric Constant and Atomic Surface Tensions. J. Phys. Chem. B 2009, 113 (18), 6378–6396. Shapiro, R.; Danzig, M., Acidic Hydrolysis of Deoxycytidine and Deoxyuridine Derivatives. General Mechanism of Deoxyribonucleoside Hydrolysis. Biochemistry 1972, 11 (1), 23–29. Cramer, C. J., Essentials of Computational Chemistry: Theories and Models. Wiley: Chichester, West Sussex, England, 2004. Suzuki, T.; Yoshida, M.; Yamada, M.; Ide, H.; Kobayashi, M.; Kanaori, K.; Tajima, K.; Makino, K., Misincorporation of 2 '-Deoxyoxanosine 5 '-Triphosphate by DNA Polymerases and Its Implication for Mutagenesis. Biochemistry 1998, 37 (33), 11592–11598. Vongchampa, V.; Dong, M.; Gingipalli, L.; Dedon, P., Stability of 2 '-Deoxyxanthosine in DNA. Nucleic Acids Res. 2003, 31 (3), 1045–1051. Norman, D. P. G.; Chung, S. J.; Verdine, G. L., Structural and Biochemical Exploration of a Critical Amino Acid in Human 8-Oxoguanine Glycosylase. Biochemistry 2003, 42 (6), 1564–1572. Norman, D. P. G.; Bruner, S. D.; Verdine, G. L., Coupling of Substrate Recognition and Catalysis by a Human Base-Excision DNA Repair Protein. J. Am. Chem. Soc. 2001, 123 (2), 359–360. Bruner, S. D.; Norman, D. P. G.; Verdine, G. L., Structural Basis for Recognition and Repair of the Endogenous Mutagen 8-Oxoguanine in DNA. Nature 2000, 403 (6772), 859–866. Yoon, J.-H.; Iwai, S.; O’Connor, T. R.; Pfeifer, G. P., Human Thymine DNA Glycosylase (TDG) and Methyl-Cpg-Binding Protein 4 (MBD4) Excise Thymine Glycol (Tg) from a Tg:G Mispair. Nucleic Acids Res. 2003, 31 (18), 5399–5404. Ocampo-Hafalla, M. T.; Altamirano, A.; Basu, A. K.; Chan, M. K.; Ocampo, J. E. A.; Cummings Jr, A.; Boorstein, R. J.; Cunningham, R. P.; Teebor, G. W., Repair of Thymine Glycol by hNTH1 and hNEIL1 Is Modulated by Base Pairing and Cis–Trans Epimerization. DNA Repair 2006, 5 (4), 444–454. Hardeland, U.; Bentele, M.; Jiricny, J.; Schär, P., Separating Substrate Recognition from Base Hydrolysis in Human Thymine DNA Glycosylase by Mutational Analysis. J. Biol. Chem. 2000, 275 (43), 33449–33456. Parker, J. B.; Stivers, J. T., Dynamics of Uracil and 5-Fluorouracil in DNA. Biochemistry 2011, 50 (5), 612–617. Rosler, A.; Panayotou, G.; Hornby, D. P.; Barlow, T.; Brown, T.; Pearl, L. H.; Savva, R.; Blackburn, G. M., The Mechanism of DNA Repair by Uracil-DNA Glycosylase: Studies Using Nucleotide Analogues. Nucleosides Nucleotides & Nucleic Acids 2000, 19 (10-12), 1505–1516. Lu, A. L.; Yuen, D. S.; Cillo, J., Catalytic Mechanism and DNA Substrate Recognition of Escherichia Coli Muty Protein. J. Biol. Chem. 1996, 271 (39), 24138–24143. Lu, A. L.; Li, X.; Gu, Y.; Wright, P. M.; Chang, D.-Y., Repair of Oxidative DNA Damage: Mechanisms and Functions. Cell Biochem. Biophys. 2001, 35 (2), 141–170. Roldan-Arjona, T.; Wei, Y. F.; Carter, K. C.; Klungland, A.; Anselmino, C.; Wang, R. P.; Augustus, M.; Lindahl, T., Molecular Cloning and Functional Expression of a Human Cdna Encoding the Antimutator Enzyme 8-Hydroxyguanine-DNA Glycosylase. Proc. Natl. Acad. Sci. U. S. A. 1997, 94 (15), 8016–8020. Fromme, J. C.; Verdine, G. L., DNA Lesion Recognition by the Bacterial Repair Enzyme MutM. J. Biol. Chem. 2003, 278 (51), 51543–51548. Slupphaug, G.; Eftedal, I.; Kavli, B.; Bharati, S.; Helle, N. M.; Haug, T.; Levine, D. W.; Krokan, H. E., Properties of a Recombinant Human Uracil-DNA Glycosylase from the Ung Gene and Evidence That Ung Encodes the Major Uracil-DNA Glycosylase. Biochemistry 1995, 34 (1), 128–138.


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84.

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27


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‡

Page 28 of 42

‡

Table 1. Comparison of the Calculated Barriers on the Potential Energy (Δ E) and Gibbs Energy (Δ G) Surfaces for the Hydrolytic Deglycosylation of Nucleoside Derivatives.

‡ a

‡ b

ΔE SN2 c

SN1

barrier

d Δmod

A

143.7

εA

SN2

barrier

d Δmod

e Δmech

0.0

148.1

0.0

136.9

–6.8

140.8

81.5

–62.2

82.7

Hx

133.7

–10.0

136.7

EA

143.0

–0.7

7DzA

171.1

G

145.8

OG X

SN1

barrier

d Δmod

barrier

4.4

143.0

0.0

132.8

–7.3

3.9

137.3

–5.7

–65.4

1.2

75.6

–67.4

–11.4

3.0

126.4

147.2

–0.9

4.2

27.4

174.3

26.2

0.0

148.3

0.0

136.9

–8.9

137.1

115.8

–30.0

7MeG

86.2

7DzG

170.9

C

nucleobase

d

Δmod

e

f

d

Δmech

Acidity

0.0

–10.2

1184.0

0.0

113.2

–19.7

–24.1

1170.0

–14.0

68.4

–64.5

–7.2

1127.6

–56.4

–16.5

117.5

–15.4

–9.0

1164.4

–19.6

142.3

–0.6

125.4

–7.5

–17.0

1180.8

–3.1

3.2

161.1

18.1

155.4

22.6

–5.7

1205.9

21.9

2.5

146.0

0.0

129.1

0.0

–16.9

1182.8

0.0

–11.2

0.2

135.2

–10.8

111.4

–17.7

–23.8

1173.1

–9.7

119.6

–28.7

3.8

119.4

–26.6

105.6

–23.4

–13.7

1151.9

–30.8

–59.6

89.0

–59.3

2.8

92.9

–53.1

71.7

–57.4

–21.2

1122.7

–60.1

25.1

174.1

25.8

3.2

170.3

24.3

156.1

27.0

–14.2

1206.7

23.9

150.5

0.0

151.2

0.0

0.7

143.4

0.0

126.2

0.0

–17.2

1197.6

0.0

U

129.2

–21.3

129.0

–22.2

–0.2

116.1

–27.3

99.5

–26.7

–16.6

1164.5

–33.1

εC

130.6

–19.9

131.3

–19.9

0.7

121.2

–22.2

102.8

–23.4

–18.5

1170.1

–27.6

T

131.4

0.0

138.3

0.0

6.9

119.3

0.0

112.6

0.0

–6.7

1168.6

0.0

Tg

151.6

20.2

161.7

23.4

10.1

143.8

24.5

124.0

11.5

–19.8

1195.5

27.0

3MeA

a

ΔG

Δmod

-1

Relative energies (kJ mol ) were obtained from IEF-PCM-B3LYP/6-311+G(2df,2p) single–point calculations on B3LYP/6-31+G(d) geometries optimized using the model depicted b

in Figure 2, and include zero-point energy corrections. Relative Gibbs energies were obtained from thermally-corrected SMD-B3LYP/6-311+G(2df,2p) single-point calculations. c

d

e

See Figure 1 for nucleoside derivatives considered. Effect of nucleobase modification calculated with respect to the corresponding canonical nucleobase. Difference in the f

barrier for the concerted and dissociative deglycosylation pathways. N9 acidity for the purine and N1 acidity for the pyrimidine derivatives. The acidity is calculated as the enthalpy of deprotonation and therefore a decrease in the deprotonation enthalpy represents an increase in the acidity.


28

Page 29 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48


‡

‡

Table 2. Comparison of the Calculated Barriers on the Potential Energy (Δ E) and Gibbs Energy (Δ G) Surfaces for the Hydrolytic Deglycosylation of Nucleoside Derivatives with Activation (Protonation) at Various Nucleobase Sites.

‡ a

‡ b

ΔE SN2 c

nucleobase

barrier

A

143.7

SN1 Δ+

d

barrier

0.0

148.1

SN2 Δ+

d

e Δmech

barrier

0.0

4.4

143.0

SN1 Δ+

d

barrier

0.0

132.8

Δ+

d

Δmech

e

Acidity

f

0.0

–10.2

1184.0

0.0

Δ+

d

A–N1H

106.2

–37.5

111.1

–37.0

4.9

107.8

–35.2

101.0

–31.8

–6.7

1147.6

–36.4

A–N3H

88.8

–54.9

90.2

–57.9

1.4

88.8

–54.2

73.9

–59.0

–14.9

1125.5

–58.5

A–N7H

76.9

–66.8

78.8

–69.3

1.9

83.1

–59.9

71.0

–61.8

–12.1

1114.7

–69.3

136.9

0.0

140.8

0.0

3.9

137.3

0.0

113.2

0.0

–24.1

1170.0

0.0

εA–N3H

84.5

–52.4

83.3

–57.5

–1.2

90.0

–47.2

91.7

–21.5

–1.7

1124.8

–45.1

εA–N7H

70.8

–66.1

75.5

–65.3

4.7

75.4

–61.9

59.6

–53.6

–15.7

1103.4

–66.6

145.8

0.0

148.3

0.0

2.5

146.0

0.0

129.1

0.0

–16.9

1182.8

0.0

92.5

–53.3

95.9

–52.4

3.4

93.2

–52.8

88.0

–41.1

–5.1

1131.0

–51.7

εA

G G–N3H G–N7H OG OG–N3H OG–O8(N7)H

g

OG–O8(N9)H

g

80.7

–65.1

87.6

–60.7

6.9

83.6

–62.4

73.7

–55.4

–9.9

1116.7

–66.1

136.9

0.0

137.1

0.0

0.2

135.2

0.0

111.4

0.0

–23.8

1173.1

0.0

124.6

–12.3

68.1

–69.0

–56.5

109.0

–26.1

79.6

–31.8

–29.5

1128.2

–44.8

61.7

–75.2

60.9

–76.2

–0.8

56.2

–79.0

61.5

–49.9

5.3

1107.2

–65.8

80.8

–56.1

87.2

–49.9

6.4

86.8

–48.4

69.7

–41.7

–17.1

1109.2

–63.9

150.5

0.0

151.2

0.0

0.7

143.4

0.0

126.2

0.0

–17.2

1197.6

0.0

75.3

–75.2

79.8

–71.4

4.5

78.8

–64.6

62.8

–63.4

–16.1

1120.3

–77.3

129.2

0.0

129.0

0.0

–0.2

116.1

0.0

99.5

0.0

–16.6

1164.5

0.0

U–O2H

41.7

–87.5

50.8

–78.2

9.1

42.7

–73.4

37.8

–61.7

–4.9

1092.6

–71.9

U–O4H

75.1

–54.1

74.8

–54.2

–0.3

83.7

–32.5

59.9

–39.6

–23.8

1117.1

–47.5

131.4

0.0

138.3

0.0

6.9

119.3

0.0

112.6

0.0

–6.7

1168.6

0.0

48.0

–83.4

54.0

–84.3

6.0

33.4

–85.9

48.2

–64.4

14.8

1096.9

–71.7

C C–O2H U

T T–O2H a

ΔG

T–O4H 76.2 –55.2 79.5 –58.8 3.3 79.6 –39.6 61.9 –50.6 –17.7 1120.3 –48.3 -1 Relative energies (kJ mol ) were obtained from IEF-PCM-B3LYP/6-311+G(2df,2p) single-point calculations on B3LYP/6-31+G(d) geometries optimized using the model depicted b

in Figure 2, and include zero-point energy corrections. Relative Gibbs energies were obtained from thermally-corrected SMD-B3LYP/6-311+G(2df,2p) single-point calculations.


29


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48

c

Page 30 of 42

d

See Figure 1 for nucleoside derivatives considered and the associated chemical numbering. Effect of nucleobase activation (protonation) calculated with respect to the e

f

corresponding unprotonated nucleobase. Difference in the barrier for the concerted and dissociative deglycosylation pathways. N9 acidity for the purine and N1 acidity for the pyrimidine derivatives. The acidity is calculated as the enthalpy of deprotonation and therefore a decrease in the deprotonation enthalpy represents an increase in the acidity. g

Orientation of the O8 proton either towards N7 (O8(N7)) or N9 (O8(N9)).


30

Page 31 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. DNA nucleobase derivatives examined in the present study, and the corresponding chemical numbering for the canonical nucleobases.


31


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 32 of 42

Figure 2. Computational model used in the present study to investigate nucleoside deglycosylation in DNA.


32

Page 33 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Reactant complexes, and concerted (SN2) and dissociative (SN1) transition states along the deglycosylation pathway for A derivatives. Select B3LYP/6-31+G(d) distances (Å) and angles (deg., in parentheses) provided including C1′···N9 (glycosidic bond length), C1′···O (nucleophilic distance) and the (∠(OC1′N9)) reaction angle.


33


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 34 of 42

Figure 4. Reactant complexes, and concerted (SN2) and dissociative (SN1) transition states along the deglycosylation pathway for G derivatives. Select B3LYP/6-31+G(d) distances (Å) and angles (deg., in parentheses) provided including C1′···N9 (glycosidic bond length), C1′···O (nucleophilic distance) and the (∠(OC1′N9)) reaction angle.


34

Page 35 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Reactant complexes, and concerted (SN2) and dissociative (SN1) transition states along the deglycosylation pathway for pyrimidine derivatives. Select B3LYP/6-31+G(d) distances (Å) and angles (deg., in parentheses) provided including C1′···N1 (glycosidic bond length), C1′···O (nucleophilic distance) and the (∠(OC1′N1)) reaction angle.


35


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 36 of 42

TOC Graphic:


36

Page 37 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 1. DNA nucleobase derivatives examined in the present study, and the corresponding chemical numbering for the canonical nucleobases. 82x180mm (150 x 150 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 2. Computational model used in the present study to investigate nucleoside deglycosylation in DNA. 82x29mm (150 x 150 DPI)


Page 38 of 42

Page 39 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 3. Reactant complexes, and concerted (SN2) and dissociative (SN1) transition states along the deglycosylation pathway for A derivatives. Select B3LYP/6-31+G(d) distances (Å) and angles (deg., in parentheses) provided including C1···N9 (glycosidic bond length), C1···O (nucleophilic distance) and the (∠(OC1N9)) reaction angle. 177x89mm (150 x 150 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Figure 4. Reactant complexes, and concerted (SN2) and dissociative (SN1) transition states along the deglycosylation pathway for G derivatives. Select B3LYP/6-31+G(d) distances (Å) and angles (deg., in parentheses) provided including C1···N9 (glycosidic bond length), C1···O (nucleophilic distance) and the (∠(OC1N9)) reaction angle. 82x134mm (150 x 150 DPI)


Page 40 of 42

Page 41 of 42

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figure 5. Reactant complexes, and concerted (SN2) and dissociative (SN1) transition states along the deglycosylation pathway for pyrimidine derivatives. Select B3LYP/6-31+G(d) distances (Å) and angles (deg., in parentheses) provided including C1···N1 (glycosidic bond length), C1···O (nucleophilic distance) and the (∠(OC1N1)) reaction angle. 82x148mm (150 x 150 DPI)



1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

88x32mm (150 x 150 DPI)


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Glycosidic Bond Cleavage in DNA Nucleosides - ACS Publications

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