Glycosidic Bond Cleavage in Deoxynucleotides: Effects of Solvent and

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Glycosidic Bond Cleavage in Deoxynucleotides: Effects of Solvent and the DNA Phosphate Backbone in the Computational Model 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 S Supporting Information *

ABSTRACT: Density functional theory (B3LYP) was employed to examine the hydrolysis of the canonical 2′-deoxynucleotides in varied environments (gas phase or water) using different computational models for the sugar residue (methyl or phosphate group at C5′) and nucleophile (water activated through full or partial proton abstraction). Regardless of the degree of nucleophile activation, our results show that key geometrical parameters along the reaction pathway are notably altered upon direct inclusion of solvent effects in the optimization routine, which leads to significant changes in the reaction energetics and better agreement with experiment. Therefore, despite the wide use of gas-phase calculations in the literature, small model computational work, as well as large-scale enzyme models, that strive to understand nucleotide deglycosylation must adequately describe the environment. Alternatively, although inclusion of the phosphate group at C5′ also affects the geometries of important stationary points, the effects cancel to yield unchanged deglycosylation barriers, and therefore smaller computational models can be used to estimate the energy associated with nucleotide deglycosylation, with the 5′ phosphate group included if full (geometric) details of the reaction are desired. Hydrogen-bonding interactions with the nucleobase can significantly reduce the barrier to deglycosylation, which supports suggestions that discrete hydrogen-bonding interactions with active-site amino acid residues can play a significant role in enzyme-catalyzed nucleobase excision. Taken together with previous studies, the present work provides vital clues about the components that must be included in future studies of the deglycosylation of isolated noncanonical nucleotides, as well as the corresponding enzyme-catalyzed reactions.

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bond cleavage in DNA (Figure 1).13−18 To this end, our computational models were specifically designed to be independent of a given active site, which permits us to focus on individual components in the proposed mechanisms of

INTRODUCTION Despite the stability of the bond connecting the nucleobases to the sugar−phosphate backbone in DNA and RNA,1 cleavage of this glycosidic linkage is an essential biological process. For example, salvage of the purines by the nucleoside hydrolases2 and phosphorylases3 and initiation of DNA repair by the DNA glycosylases4,5 rely on this reaction, to name a few fundamental examples. The enzymes catalyzing these vital processes have all been proposed to rely on active-site residues to assist nucleobase departure (through either hydrogen-bonding interactions or protonation of the base),6−8 stabilize the formation of a cationic charge on the sugar moiety,9,10 and activate the (water, active-site amine, or phosphate) nucleophile that attaches to the sugar and displaces the base.11,12 Although it is agreed that these features are essential for reducing the otherwise high barrier associated with glycosidic bond cleavage, the relative importance of these elements and their exact mechanistic role are often unclear,4,5 and depend on the nature of the substrate (e.g., natural versus damaged base, ribose versus deoxyribose sugar, nucleoside versus nucleotide) and/or the enzyme under consideration. To fully appreciate the individual contributions of the various features involved in enzymatic depurination or depyrimidination, work in our group has used computational chemistry to characterize the kinetics and thermodynamics of glycosidic © XXXX American Chemical Society

Figure 1. Structure and atomic numbering of the nucleobases (uracil (U), adenine (A), cytosine (C), guanine (G), and thymine (T)), as well as the sugar backbone models considered in the present study (R = CH3 or [PO3CH3]−Na+, and R′ = CH3). Received: September 28, 2012 Revised: November 13, 2012

A

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small model studies, we focus our efforts on the hydrolysis of the canonical 2′-deoxynucleotides due to the abundance of water in nature and the anticipated role of water in the glycosidic bond cleavage facilitated by several enzymes.4,5,11 We acknowledge that the uncatalyzed hydrolysis reaction,15,17,67,68 and some enzyme-catalyzed reactions (e.g., repair of uracil by hUNG2),8,69 may proceed via an alternative (dissociate (SN1)) pathway. However, we only consider the concerted (SN2) pathway for glycosidic bond cleavage to allow direct comparison among the canonical nucleotides and to previous studies in the literature.13,14,18 Furthermore, this pathway is particularly consistent with experimental evidence since excision of some nucleobases (for example, thymine by thymidine phosphorylase) has been suggested to be concerted or only slightly asynchronous.5,70 Previous computational studies of the structure (e.g., preferred conformation) of 2′-deoxyguanosine 5′-monophosphate,49,51,54,57,60 and select dinucleoside 5′-monophosphates,55,58,61,63,64 report that solvent effects must be directly taken into account in the optimization routine in order to characterize DNA-relevant geometries. Since previous studies on DNA nucleotide models without the phosphate group (R = CH3, Figure 1) were performed in the gas phase with the effects of the surrounding environment at best included in implicit single-point calculations,13,14,16,18,23−26,28−33,35−38 the present study begins by determining the effects of the surrounding (water) environment on the structure of stationary points along the reaction pathway, as well as the associated changes in the reaction energetics, for the equivalent (R = CH3) small model. Subsequently, the effects of the 5′-phosphate group on the reaction energetics in water are evaluated using an expanded computational model (R = [PO3CH3]−Na+, Figure 1). Finally, the effects of the 5′-phosphodiester moiety on the ability of hydrogen bonding with the nucleobase to decrease the barrier heights and enhance the reaction exothermicity are examined because of the anticipated importance of nucleobase−amino acid interactions in enzyme-catalyzed reactions. When combined with findings from our previous work,13,14,18 the present study will aid the identification of the most computationally efficient approach for studying the deglycosylation of DNA nucleotides. These findings can be used in future investigations of unnatural systems. For example, information regarding the relative stability of the glycosidic bond in natural and modified nucleobases is necessary to understand the detrimental effects of some forms of DNA damage.14,18,22,24,25,29 In addition to providing the basis (uncatalyzed reaction) for understanding the associated enzyme reaction, this work will reveal key features (including identification of truncation points and species that must be treated with high levels of theory) that should be carefully considered when designing models for large-scale calculations on enzymes that catalyze deglycosylation of DNA nucleotides, including those involved in DNA repair or nucleotide salvage pathways.

nucleobase excision enzymes. By studying the hydrolysis of 2′deoxyuridine,13 our initial work revealed important insights into the effects of nucleophile activation and interactions with the (uracil) nucleobase on the concerted (SN2) reaction. A followup study on the excision of the canonical nucleobases from 2′deoxynucleosides (R = R′ = CH3) exposed the effects of the nucleobase and the energetic effects of the (bulk) environment, and clarified the role of hydrogen-bonding contacts with the nucleobase, on the hydrolytic glycosidic bond cleavage.14 By expanding the computational model to include active-site amine nucleophiles and an oxidatively damaged purine,18 we subsequently provided a greater understanding of the relative effects of the nucleophile, damage to the nucleobase, and nucleobase orientation with respect to the sugar moiety on the concerted deglycosylation pathway. These studies complement other work in the literature that typically focused on the effect of charge (for example, protonation, hydrogen bonding, or metal binding) on glycosidic bond stability, either implicitly through measuring changes in acidities19−22 or glycosidic bond length,23−26 or explicitly through barrier height determination.27−31 Additional small model studies have included features of particular enzyme active sites, for example, hOgg1,32−34 hUNG2,35 hNEIL,36,37 and hTDG.38 Large model studies of enzymatic deglycosylation have combined the various small model observations to gain further insight into how the different features work together.39−46 Combined, this body of research has provided qualitative explanations for the relative base excision rates observed in biological systems. Despite the crucial information obtained from the previous studies of the glycosidic bond cleavage in DNA outlined above, the computational model adopted must be more closely examined. Specifically, the majority of previous high-level (small model) computational studies have used models that replace the phosphate backbone with a hydrogen or methyl group (i.e., R = H or CH3, Figure 1). Indeed, studies featuring nucleotide models that explicitly include the phosphodiester moiety are relatively scarce in the literature regardless of whether reactions34,47,48 or simply the structures49−64 of these DNA components are examined. Experimentally, however, the measured rates for base excision from the free nucleosides in solution1 differ from those reported for double-stranded DNA.65,66 Furthermore, while many factors could be playing a role (phosphate backbone, hydrogen bonding, nucleobase stacking, acid catalysis, and solvent accessibility of C1′), there is currently no definitive explanation for the difference in excision rates. Therefore, new studies are required to evaluate the effect of different components of the DNA environment on the glycosidic bond stability, as well as the ability of small computational models to afford quantitative information about the deglycosylation reactions occurring in larger biosystems. Additional studies can also direct future large model studies by, for example, specifying the components that must be included, be described with high-level methods, and/or be free to optimize in the model. The goal of the present work is to determine whether the inclusion of a 5′-phosphodiester group affects the calculated glycosidic bond stability, and therefore the structural and energetic features associated with the deglycosylation of 2′deoxynucleotides in DNA. Indeed, this negatively charged moiety may affect the DNA deglycosylation reaction by destabilizing the departing (often negatively charged) nucleobase4−6 or stabilizing the cationic charge developing on the sugar moiety,9,41 as a few examples. As done in our previous

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COMPUTATIONAL DETAILS All geometries were initially taken from our previous gas-phase study14 and optimized with the B3LYP functional and the 631G(d) basis set. The relative energies were subsequently obtained from B3LYP/6-311+G(2df,p) single-point calculations. Previous work in our laboratory shows that 6-31G(d) geometries yield relative energies obtained from further singlepoint calculations in excellent agreement with geometries B

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Figure 2. Selected B3LYP/6-31G(d) bond lengths (angstroms) (and angles (degrees), in parentheses) in the reactant, transition state, and product complexes for the deglycosylation of 2′-deoxyuridine facilitated by OH− (left) and HCOO−···H2O (right) for the R = CH3 (optimized in gas13 (top) and water (middle)) and R = [PO3CH3]−Na+ (bottom) truncated nucleotide models.

Figure 3. Selected B3LYP/6-31G(d) bond lengths (angstroms) (and angles (degrees), in parentheses) in the reactant, transition state, and product complexes for the hydrolytic deglycosylation of 2′-deoxyadenine facilitated by OH− (left) and HCOO−···H2O (right) for the R = CH3 (optimized in gas13 (top) and water (middle)) and R = [PO3CH3]−Na+ (bottom) truncated nucleotide models.

states, intrinsic reaction coordinates (IRC) were followed to confirm stationary points are connected along the reaction pathway. Scaled (0.9806) zero-point energy corrections were added to all relative energies. All calculations were performed with the Gaussian 09 program suite.71

obtained using larger (6-31+G(d,p)) basis sets for the hydrolysis of 2′-deoxyuridine.13 Furthermore, despite the charge associated with the phosphate backbone, the structures of the dinucleoside monophosphates are largely unaffected by the use of more computationally efficient basis sets void of diffuse functions.60,63 Bulk solvation (water) effects were taken into account using the IEF-PCM implicit solvation model (ε = 78.36) in either single-point calculations on gas-phase geometries or during both the geometry optimization step and energy calculations. Following characterization of the transition

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RESULTS AND DISCUSSION

In enzyme-catalyzed nucleotide hydrolysis pathways, it is almost uniformly proposed that the water nucleophile is activated by an active-site amino acid residue.2,4,5,11 ComputaC

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Table 1. Comparison of the Calculated Barrier and Reaction Energy (kJ mol−1) for the Hydrolytic Deglycosylation of 2′Deoxynucleotides (R = CH3, Figure 1) by the OH− or HCOO−···H2O Nucleophile in Different Environmentsa gas//gasb nucleotide

barrier

water//gasc

reaction energy

barrier

Δsolve

water//waterd

reaction energy

Δsolve

barrier

Δoptf

reaction energy

Δoptf

81.5 92.3 88.3 84.1 81.1

84.3 99.8 104.2 103.2 88.7

11.3 10.7 16.5 12.4 15.6

−109.0 −99.7 −79.2 −96.4 −109.0

−27.1 −40.7 −30.5 −29.6 −29.1

3.7 3.1 6.2 1.8 2.8

131.1 144.8 159.4 147.0 135.2

22.5 21.4 25.8 19.6 22.6

43.0 53.3 74.9 55.1 47.2

23.4 24.5 27.8 18.3 30.4

−

dU dA dC dG dT

49.9 61.0 64.9 60.5 51.6

−163.4 −151.3 −137.0 −150.9 −161.0

73.0 89.1 87.7 90.8 73.1

23.1 28.1 22.8 30.3 21.5

dU dA dC dG dT

108.1 122.2 130.4 127.0 110.3

15.9 25.7 40.9 35.0 14.0

108.6 123.4 133.6 127.4 112.6

0.5 1.2 3.2 0.4 2.3

OH −81.9 −59.0 −48.7 −66.8 −79.9 HCOO−···H2O 19.6 28.8 47.1 36.8 16.8

a

Energies obtained from (PCM-)B3LYP/6-311+G(2df,p) single-point calculations on (PCM-)B3LYP/6-31G(d) geometries and include scaled (0.9806) zero-point vibrational energy corrections. bGas-phase structures and gas-phase energies adopted from ref 13. cGas-phase structures with solvent-phase single-point energies. dSolvent-phase structures with solvent-phase single-point energies. eDifference between solvent-phase and gasphase single-point energies obtained using the gas-phase structures. fDifference between the solvent-phase single-point energies obtained using structures optimized in water and the gas phase.

the nucleophile and the departing nucleobase. In the product complex, a bond is fully formed between the nucleophile and the sugar moiety, while the base anion is stabilized through hydrogen-bonding interactions with the sugar−nucleophile complex. The highly charged and reactive OH− nucleophile affords tight transition states and small barriers (50−65 kJ mol−1), as well as significantly exothermic reactions (−135 to −165 kJ mol−1, Table 1). In contrast, the greater stability of the HCOO−···H2O nucleophile in the reactant complex, as well as the decreased ability of this nucleophile to stabilize the positive charge forming on the sugar moiety in the transition state, results in much later transition states with glycosidic bond lengths over 2.5 Å. As a result, the barriers for glycosidic bond cleavage by HCOO−···H2O are large (110−130 kJ mol−1) and the reactions are endothermic (by 15−40 kJ mol−1, Table 1). The effects of the surrounding environment on chemical reactions are most commonly estimated using implicit solventphase (PCM) single-point calculations on gas-phase optimized structures.72 When this approach is applied to the hydrolysis of the R = CH3 nucleotide model, the barrier height increases and the reaction exothermicity decreases for both nucleophiles (Δ solv , Table 1). Specifically, the barriers are up to approximately 30 kJ mol−1 larger for the OH− nucleophile and only slightly larger (up to 3 kJ mol−1) for HCOO−···H2O, while the reaction is less exothermic by up to approximately 90 and 6 kJ mol−1 for the two nucleophiles, respectively. These changes arise because of ground state (reactant) stabilization resulting from a decrease in the power of the nucleophile (stabilization of the nucleophile) in the reactant complex. Although reactant stabilization is likely the most important factor, this may at least in part be complemented by changes in the stabilization of the charges forming on the sugar moiety and nucleobase over the course of the reaction. The effect of these factors is larger when the reaction is catalyzed by the hydroxyl anion due to the greater concentration of charge in this nucleophile compared with HCOO−···H2O. These calculated solvent effects are in accord with changes in the reaction energetics for hydrolysis by OH − compared with HCOO−···H2O. Specifically, the more stabilization provided to the nucleophile in the reactant (through either discrete

tional studies have also emphasized the important role of nucleophile activation in DNA deglycosylation reactions.13,15,17,30 Indeed, barriers ranging from direct hydrolysis (by a single water molecule) to hydrolysis by a fully activated (deprotonated) water can be obtained depending on the acidity of the molecule used to activate (deprotonate) the nucleophile.13 Therefore, as done in previous studies by our group,13,14,35 two representative nucleophiles are considered in the present work, namely, the hydroxyl anion (OH−), which represents full water activation, and partial activation by the formate anion (HCOO−···H2O). Using these nucleophiles, we consider the effects of the surrounding environment and the DNA 5′-phosphodiester backbone on the structures of all stationary points along the SN2 reaction coordinate, as well as the associated reaction energetics. Subsequently, the effects of both factors on the ability of hydrogen-bonding interactions between small molecules and the nucleobase to lower the deglycosylation barrier will be considered due to the proposed role of active-site interactions in stabilizing the departing base in enzyme-catalyzed reactions. Effects of Solvation. Previous work in our laboratory has emphasized that replacing the phosphodiester backbone at C5′ and C3′ with a hydroxyl group (R = R′ = H, Figure 1) inadequately models deglycosylation of DNA nucleotides because of interactions between the hydroxyl and the nucleobase or nucleophile.13 Instead, we reported that methoxyl groups (R = R′ = CH3) afford more accurate models. Therefore, in the present study, R′ = CH3 for all models, and the smallest nucleotide model considered also assigns R = CH3. Structural details of the gas-phase hydrolysis for all nucleotides and nucleophile combinations considered in the present work are provided in the Supporting Information (Tables S1−S3). Since the reaction pathways are similar for all bases, those for excision of uracil and adenine are presented as representative examples in Figures 2 and 3, respectively. In all reactions, the nucleophile is initially complexed to the nucleotide through hydrogen-bonding interactions with the sugar moiety and moves closer to C1′ in the transition state. The formation of a partial positive charge on the sugar moiety in the transition state is stabilized by negative charges on both D

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0), which is in contrast to the greater exothermicity found when OH− is the nucleophile (Δopt < 0). The above results illustrate that key geometrical parameters in important species along the reaction pathway can significantly change upon inclusion of (implicit) solvent in the optimization routine, which in turn affects the reaction energetics. Furthermore, solvent-phase single-point calculations on gas-phase structures cannot recover these features. Indeed, we note that the trend in the hydrolysis barrier with respect to the nucleobase obtained from solvent-phase single-point calculations (G ≈ A > C > T ≈ U for OH− and C > G > A > T > U for HCOO−···H2O) differs from that obtained from solvent-phase optimizations (C > G > A > T > U for both nucleophiles). Furthermore, this trend is in better agreement with the trend in experimentally measured excision rates for free nucleosides1 and in DNA,65,66 especially for the pyrimidines. These findings emphasize the importance of performing solvent-phase calculations when examining the hydrolysis of the canonical nucleotides. This conclusion likely extends to the hydrolysis of damaged nucleotides, as well as modified nucleobases used in biotechnological applications. Furthermore, these results highlight the importance of designing computational models that accurately describe the complete active-site environment when modeling enzymecatalyzed hydrolysis of natural or damaged nucleotides. Effects of Phosphate Model. The previous section, Effects of Solvation, emphasizes the importance of including the environment when examining hydrolytic excision of a nucleobase from DNA. As discussed in the Introduction, differences in the base excision rate from nucleosides versus double-stranded DNA suggests that the charged phosphate backbone may be one of the factors that affects the reported structures and energetics for DNA deglycosylation.1,65,66 Nevertheless, most computational studies on the structure or reactions involving DNA employ a nucleoside model (R = CH3 or smaller).13,14,16,18,22−26,28−33,35−38 Furthermore, larger computational models previously used in the literature that include the 5′-phosphate group may be inappropriate for the present study. For example, some studies have employed constraints on the phosphate backbone model to prevent the formation of structures unrealistic to the DNA environment,73 which may be inaccurate when studying the nucleotide deglycosylation since changes to the backbone geometry may occur throughout reaction pathways. Alternatively, some studies have used dinucleoside 5′-monophosphate models,48,55,58,60,61,63,64 which can introduce sequence dependencies that are beyond the scope of the present work. In the present study, the effects of the phosphate group are taken into account by representing the C5′ phosphate as R = [PO3CH3]−Na+ (Figure 1). This model includes a methyl cap to avoid interactions between the nucleobase and the backbone along the reaction pathway that are non-native to DNA without invoking constraints on the structure of the backbone, and a sodium counterion to neutralize the negative charge of the backbone, which was previously shown to provide the most accurate structural representations of DNA nucleotides.60 As for the smaller (R = CH3) model considered in the present work, the phosphodiester backbone at C3′ is replaced with a methoxyl group (R′ = CH3) to prevent interactions with the nucleophile that are non-native to the DNA environment.13 Upon extension of the computational model to include the 5′-phosphate residue as described above, significant structural changes occur throughout the reaction pathway (see Tables

hydrogen-bonding interactions or implicit solvation), the larger the reaction barrier and the more endothermic the overall reaction. The above discussion coupled with previous computational studies15,17 illustrates that inclusion of (bulk) solvent effects can significantly alter the calculated energetics for DNA nucleotide hydrolysis. Therefore, we subsequently considered the effects of solvent-phase optimizations (Δopt) on the structures (Figures 2 and 3 and Figures S1−S4 in the Supporting Information) and energetics (Table 1) of nucleotide hydrolysis described using the R = CH3 model (Figure 1). The structural and resulting energetic changes upon optimization in water are slightly different for the two nucleophiles under consideration. In the case of OH−, the most notable structural changes include increases in the nucleophile distance in the reactant (by 0.12− 0.27 Å, Table S2 in the Supporting Information) and the glycosidic bond length in the transition state (by approximately 0.06 Å, Table S1 in the Supporting Information) due to enhanced stabilization of the negative charge on the nucleophile and departing base, respectively. The decreased nucleophilicity of OH − and the later transition state cooperatively increase the barrier by 10−15 kJ mol−1 compared with solvent single-point calculations on gas-phase structures (Δopt, Table 1). Although the relative orientation of the nucleobase with respect to the sugar moiety observed in the gas phase is maintained in the reactant and transition state upon optimization in water, there are large changes in χ (∠(O4′C1′N1C1) in the pyrimidines and ∠(O4′C1′N9C4) in the purines) in the product complexes (by up to 70−100°, Table S3 in the Supporting Information). These differences arise since χ adjusts over the course of the gas-phase reaction in order to stabilize the base anion through hydrogen-bonding interactions with the sugar moiety. In contrast, χ is maintained at nearly constant values throughout the reaction in water since the solvent adequately stabilizes the base anion. Indeed, stabilization provided to the departing base by the solvent leads to more exothermic reaction energies compared with solvent-phase single-point calculations on gas-phase structures (Δopt < 0, Table 1). Nevertheless, the stabilizing effects of solvent lead to overall reaction energies that are less exothermic than those observed for the gas-phase dissociation. Similar to OH− catalyzed base departure, inclusion of (bulk) solvent while optimizing the stationary points for hydrolysis by HCOO−···H2O notably increases the glycosidic bond length in the transition state (by up to 0.03 Å, Table S1 in the Supporting Information). However, unlike the OH− catalyzed reaction, the nucleophilic distance significantly increases in both the reactant (by up to 0.14 Å, Table S2 in the Supporting Information) and transition state (by up to 0.10 Å). This suggests that the nucleophilicity of HCOO−···H2O is reduced in water, which is further supported by decreased proton transfer from water to HCOO− in the solvent-phase transition states. The looser transition states increase the barrier by up to 26 kJ mol−1 (Table 1). Therefore, although the solvent affects the nucleophilic strength of both HCOO−···H2O and OH−, the effects are larger for HCOO−···H2O. This situation partially arises since the solvent weakens the hydrogen bond between the formate anion and water, which leads to less activation of the nucleophile. In contrast, since OH− is inherently a stronger nucleophile, it maintains significant nucleophilic power in solution. These factors lead to an up to 30 kJ mol−1 more endothermic HCOO−···H2O reaction pathway in water (Δopt > E

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S1−S3 in the Supporting Information, Figures 2 and 3, and Figures S1−S4 in the Supporting Information), which are consistent across all nucleotides considered in the present work. In the case of the OH− nucleophile, large changes occur in the reactant complexes, which exhibit decreases in the glycosidic bond (by up to 0.03 Å) and nucleophile (by up to 0.5 Å) distances. The relative orientation of the nucleobase and sugar moiety also changes significantly in the purine reactant complexes, where the sugar puckering changes to O4′-endo and therefore χ increases by 30−35° due to loss of the C8− H···O5′ hydrogen bond. In contrast, the transition structures do not appreciably change upon model expansion. The product complexes contain much longer glycosidic (C1′···N1(N9)) distances (by up to 0.8 Å) in the phosphate model due to different interactions at the phosphate group. As discussed for the OH− nucleophile, the glycosidic bond length in the reactant associated with the HCOO−···H2O facilitated deglycosylation pathway decreases by up to 0.02 Å. However, in the case of the HCOO−···H2O mediated reaction, the nucleophile distance in the reactant increases by up to 0.14 Å, and is accompanied by a change in the reactant sugar puckering from the C3′-endo conformation present in all other structures considered to C2′-endo. These factors are accompanied by significant alterations to the transition structure, where the glycosidic distance decreases by up to 0.1 Å, the nucleophile distance increases by up to 0.2 Å, and the sugar puckering changes from C3′-endo to C2′-endo. As discussed for the OH− nucleophile, the product complexes of the R = [PO3CH3]−Na+ model have much longer glycosidic bond lengths (by up to 0.3 Å) and the sugar puckering is uniformly C2′-endo. The majority of these geometric changes for the HCOO−···H2O reaction pathway are likely the result of a large change in the nucleophile orientation (i.e., formate anion is directed toward O4′ rather than C3′ of the sugar moiety). Interestingly, both conformations can be characterized for the R = CH3 model (Tables S4 and S5 and Figure S5 in the Supporting Information), which have very similar reaction energetics despite these geometrical differences. Even though there are significant structural differences in the reactant and product complexes for the OH− catalyzed deglycosylation, the calculated barriers change by less than 2 kJ mol−1 and the overall reaction energies change by less than 6 kJ mol−1 upon addition of the 5′-phosphate group (Δphos, Table 2). Similarly, changes in the HCOO−···H2O mediated reaction barriers and energetics are less than 8 and 6 kJ mol−1, respectively (Δphos, Table 2). Thus, it appears that any structural effects and electrostatic effects (the anionic charge provides stabilization of the (partially) cationic sugar moiety and destabilization of the nucleobase) due to the phosphate group cancel along the reaction pathway to yield no overall change in the reaction energetics. In summary, the inclusion of the 5′-phosphate residue in solvent-phase optimizations does not change the (solventphase) reaction energetics for either nucleophile considered in the present work. Therefore, if the sole objective of the computational study is to evaluate the energetics of the reaction, then a reduced computational model (with a methoxyl group replacing the phosphodiester backbone) can be used without loss of accuracy. However, the effects of the phosphate group on the structures of the stationary points along the reaction pathway can be significant. Therefore, the accuracy of the small model must be due to a cancellation of effects and it is unclear whether this cancellation will consistently occur for

Table 2. Effects of the 5′-Phosphate Group on the Calculated Barrier and Reaction Energy (kJ mol−1) for the Hydrolytic Deglycosylation of Nucleotides by the OH− or HCOO−···H2O Nucleophilea R = [PO3CH3]−Na+ b

R = CH3b nucleotide

barrier

dU dA dC dG dT

84.3 99.8 104.2 103.2 88.7

dU dA dC dG dT

131.1 144.8 159.4 147.0 135.2

reaction energy

barrier

OH− −109.0 82.1 −99.7 101.9 −79.2 106.5 −96.4 104.2 −109.0 87.0 HCOO−···H2O 43.0 127.1 53.3 144.3 74.9 151.0 55.1 147.0 47.2 132.8

Δphosc

reaction energy

Δphosc

−2.2 2.0 2.3 1.0 −1.7

−113.1 −93.7 −77.8 −91.9 −107.0

−4.1 6.0 1.4 4.5 2.0

−4.0 −0.5 −8.4 0.0 −2.4

44.5 59.3 74.8 62.4 48.1

1.5 6.0 −0.0 7.3 0.9

a

Energies obtained at the PCM-B3LYP/6-311+G(2df,p)//PCMB3LYP/6-31G(d) level of theory and include scaled (0.9806) zeropoint vibrational energy corrections. bSee Figure 1 for model definition. cDifference between the energies obtained with the R = [PO3CH3]−Na+ and R = CH3 nucleotide models.

noncanonical nucleotides. Furthermore, if full details of the reaction potential energy surface are required, then the effects of including the 5′-phosphate residue in the computational model must be carefully considered. It may also be necessary to directly include the 5′-phosphodiester backbone when considering deglycosylation of modified nucleobases. For example, models that accurately describe the DNA backbone may be especially important when studying DNA nucleotides with increased bulk, such as the widely discussed damaged purine adducts with bulky substituents at the C8 position (for example, damage by aromatic amines,74,75 or phenolic compounds76,77), which may result in close base−phosphate contacts. Effects of Hydrogen-Bonding Interactions with the Nucleobase. As mentioned in the Introduction, stabilization of the departing nucleobase through either full protonation or hydrogen-bonding interactions (partial protonation) is an important proposed function of enzymes that catalyze nucleotide deglycosylation.6−8 Therefore, previous work in our group has investigated the extent to which hydrogenbonding interactions with small molecules spanning a range in acidity (XH = HF, H2O, and NH3) enhance the leaving ability (acidity) of the natural, as well as a variety of damaged, nucleobases.19−21,35,78,79 We calculated that the gas-phase acidity can be increased by up to 60 kJ mol−1, and the effect is still significant (30 kJ mol−1) upon inclusion of (implicit) solvent (water) through single-point calculations.78 The conclusions for the small molecules also hold upon consideration of hydrogen-bonding interactions with amino acid fragments,21 and the resulting estimated effects show strong correlations with experimentally observed effects on the deglycosylation rates. For example, Raman IR estimates the effect of an Nε−H···O2 hydrogen bond between a histidine residue and uracil to contribute ∼20−24 kJ mol−1 to the catalytic effect of UDG.6 In the presence of bulk (ether) solvent, the calculated change in N1 acidity of uracil due to this kind of interaction is 24.3 kJ mol−1.21 Furthermore, subsequent F

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work estimated the effects of XH on the deglycosylation reaction energetics to be up to 30 kJ mol−1 depending on the nucleophile considered, which dictates the tightness of the transition state.13 Although the results discussed in the previous paragraph suggest that hydrogen-bonding interactions can significantly catalyze the glycosidic bond cleavage in DNA, this finding must be reevaluated in light of the previous sections, which suggest that optimizations in water and inclusion of the 5′-phosphate residue may alter the reaction energetics or structures of species involved in nucleotide deglycosylation pathways. Due to the similarities in these effects for all nucleotides (Tables S1−S3 and Figures S1−S4 in the Supporting Information), we only consider the effects of hydrogen-bonding interactions on the deglycosylation of 2′-deoxyuridine (as shown in Figure 2), which has shown to be an exemplary test case.13−15,17 The hydrogen-bonding sites considered are displayed in Figure 4, where the small molecules generally form bidentate hydrogen bonds with uracil and our notation indicates the uracil hydrogen-bond acceptor, with the donor in parentheses.

Table 3 compares the effects of hydrogen-bonding interactions (ΔXH) on the deglycosylation reaction energetics in water calculated using the structures obtained for the R = CH3 model in the gas phase or water, as well as the structures obtained for the larger model that includes the phosphate group (R = [PO3CH3]−Na+). As expected, ΔXH on the barrier in water decreases with the acidity of the small molecule bound (HF > H2O > NH3) regardless of the environment (gas or water) or model (R = CH3 or [PO3CH3]−Na+) used to optimize the geometries. Specifically, hydrogen fluoride leads to the largest stabilization, while ammonia sometimes even increases the barrier heights. Additionally, ΔXH on the barrier decreases as the power of the nucleophile increases (HCOO−···H2O > OH−). Specifically, as the nucleophile becomes more powerful, the transition state occurs earlier (shorter glycosidic distance), which results in less charge on the nucleobase and less stabilization of the departing base by the small molecule. In contrast to the barriers, ΔXH on the overall reaction energies is nearly independent of the nucleophile for the R = CH3 model since all product complexes involve the uracil anion bound to the sugar−nucleophile complex and therefore lead to a similar charge on the nucleobase. In the case of the R = [PO3CH3]−Na+ model, ΔXH for the reaction energies displays greater variations since the product complexes sometimes exhibit strong interactions between XH and the phosphate backbone. Since it is difficult to isolate the effects of the hydrogen-bonding interactions between the nucleobase and the small molecule on the associated reaction energies in these structures, we focus our discussion on the reaction barriers, where all transition states are void of interactions involving the small molecule and the phosphate backbone.

Figure 4. Binding sites of small molecules (XH = HF, H2O, or NH3) to uracil considered in the present study.

Table 3. Effects of Hydrogen-Bonding Interactions between Small Molecules (XH = HF, H2O, or NH3) and Uracil on the Calculated Barrier and Reaction Energy (kJ mol−1) for the Hydrolytic Deglycosylation of dU by the OH− or HCOO−···H2O Nucleophilea,b R = [PO3CH3]−Na+

R = CH3 model geometry O2(N3)

O4(N3)

water//gas barrier

ΔXHe

c

reaction energy

water//water ΔXHe

barrier

ΔXHe

d

water//waterd

reaction energy

ΔXHe

barrier

ΔXHe

reaction energy

ΔXHe

−109.0 −139.8 −130.3 −120.7 −118.1 −103.3 −104.8

−30.8 −21.3 −11.7 −9.0 5.7 4.2

82.1 69.1 71.5 78.6 80.8 94.9 91.3

−13.0 −10.7 −3.5 −1.3 12.8 9.2

−113.1 −131.3 −133.7 −136.2 −142.7 −97.2 −99.3

−18.2 −20.6 −23.1 −29.6 15.9 13.8

43.0 15.6 22.1 35.9 34.7 50.1 48.6

−27.4 −20.9 −7.1 −8.3 7.1 5.6

127.1 106.9 112.5 122.1 124.9 140.4 138.1

−20.2 −14.6 −5.0 −2.2 13.3 11.0

44.5 16.5 26.1 36.7 41.0 56.6 56.0

−28.1 −18.4 −7.8 −3.5 12.1 11.5

−

HF HF H2O H2O NH3 NH3

HF HF H2O H2O NH3 NH3

73.0 58.5 60.0 68.1 69.1 76.8 76.9 108.6 84.5 91.1 101.3 105.6 112.1 111.9

−14.5 −13.0 −4.9 −3.9 3.8 3.9

−81.9 −107.1 −100.4 −89.4 −86.1 −77.8 −76.9

−25.2 −18.5 −7.5 −4.2 4.1 5.0

−24.1 −17.5 −7.3 −3.0 3.5 3.3

19.6 −9.7 −3.6 10.2 15.8 25.8 23.4

−29.3 −23.2 −9.4 −3.8 6.2 3.8

OH 84.3 69.4 −14.8 73.8 −10.5 81.3 −3.0 81.9 −2.4 94.6 10.3 91.5 7.2 HCOO−···H2O 131.1 107.2 −23.9 115.6 −15.5 124.8 −6.3 127.8 −3.3 141.6 10.5 140.6 9.5

a

See Figure 1 for model definitions and Figure 4 for definition of uracil binding sites. bRelative energies were calculated at the PCM-B3LYP/6311+G(2df,p)//(PCM-)B3LYP/6-31G(d) level of theory. cGas-phase structures were obtained from ref 13. dSolvent-phase structures. eΔXH is calculated as the difference between the barrier or reaction energy calculated with and without a small molecule bound to uracil obtained using the same computational approach. G

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otherwise chemically modified nucleotides should take careful measures to account for the environment either through the use of (implicit) solvent-phase calculations or through the inclusion of a sufficient number of active-site residues in the case of enzyme modeling. In addition to solvation, the effects of the negatively charged phosphodiester in the DNA backbone were closely considered. Although expansion of the computational model to include the phosphate backbone can affect the geometry of important stationary points for nucleotide deglycosylation, the overall energetics can be reliably reproduced with a smaller computational model, including the effects of hydrogen-bonding interactions on the glycosidic bond cleavage barrier. Thus, computational models that replace the phosphate backbone with a methyl group deliver a good compromise between accuracy and computational efficiency when the relative barriers for the hydrolysis of different nucleotides are desired. Future work must determine whether these results hold for other DNA nucleotides, including various forms of damage, which may exhibit different interactions with the 5′-phosphate group, and other nucleophiles, which can have different nucleophilic powers and/or different associated deglycosylation mechanisms.

The effects of hydrogen bonding on the 2′-deoxyuridine deglycosylation barrier are generally only slightly smaller when optimizations are performed in water rather than in the gas phase (see ΔXH for R = CH3, Table 3). This result is perhaps contrary to that anticipated based on the representative structures displayed in Figure 2. Specifically, optimization in water lengthens the glycosidic bond, which should increase the negative charge on the departing base in the transition state and afford a larger ΔXH. However, discrete hydrogen-bonding interactions are weaker in water than in the gas phase, where the hydrogen−bond lengths to O2 and O4 generally increase and those to the N3−H basic site decrease upon inclusion of solvent in the optimization. Overall, the effects of a longer glycosidic bond length and weaker hydrogen bonds with XH in water cancel to yield a net zero change in ΔXH. Similarly, despite structural changes upon model extension to include the 5′-phosphate group, the effects due to changes in the reactant and transition state structures, as well as the hydrogen bonding between XH and uracil, sum to yield similar ΔXH values (Table 3). It should also be noted that the magnitude of the barriers and reaction energies are very similar for the R = CH3 and R = [PO3CH3]−Na+ models regardless of the small molecule bound (Table 3), which reemphasizes the ability of the smaller model to accurately reproduce relative energies when optimizations are performed in (implicit) solvent. In summary, the calculated effects of hydrogen-bonding interactions between the nucleobase and small molecules are similar regardless of whether the (bulk) solvent (water) is included in the optimization routine or the 5′-phosphate group is included in the solvated model. This suggests that reduced computational models can be used to evaluate the role of base stabilization via discrete interactions in enzymatic deglycosylation reactions. Most importantly, the effects of hydrogenbonding contacts on the reaction energies are significant (up to 30 kJ mol−1 depending on the nucleophile). Furthermore, previous calculations with the same small molecule bound at both uracil sites considered in the present work show that even greater reductions in barrier heights occur when more than one small molecule is simultaneously bound to the nucleobase.13,14,35 This conclusion also holds true when a phosphate group is included in the computational model (Table S6 in the Supporting Information). Therefore, our results support previous suggestions that enzymes can exploit hydrogen bonding between the nucleobase and active-site residues to at least in part catalyze biologically important deglycosylation reactions, and they suggest that such substrate−enzyme interactions must be accurately portrayed when modeling enzyme-catalyzed reactions.

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ASSOCIATED CONTENT

S Supporting Information *

Changes in the glycosidic bond and nucleophile distances (Tables S1 and S2), as well as χ dihedral angles (Table S3), upon inclusion of solvent and the phosphate backbone; the complete reaction pathways for all nucleotides in solvent for the two models considered in the present work (Figures S1−S4); representative alternative orientation of the HCOO−···H2O nucleophile in water (Figure S5), as well as the associated geometries and energies (Tables S4 and S5); and representative effects of more than one hydrogen-bonding interaction on the deglycosylation energetics (Table S6). Full citation for ref 71. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel.: (403) 329-2323. Fax: (403) 329-2057. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This research was supported by the Natural Sciences and Engineering Research Council (NSERC), the Canada Research Chair program, and the Canada Foundation for Innovation (CFI) for S.D.W., an NSERC graduate student scholarship (CGS-D) for J.L.K., and a Chinook Summer Research Award from the University of Lethbridge for S.A.P.L. Calculations were conducted on the Up-scale and Robust Abacus for Chemistry in Lethbridge (URACIL) computing cluster.

■

CONCLUSIONS The present work has carefully improved upon the computational approach previously used in the literature to characterize the hydrolytic deglycosylation of nucleotides in DNA, a fundamental reaction in biological systems. In the first instance, the (bulk) effects of a fully solvated (water) environment were directly included in the optimization routine. For all nucleotides and nucleophiles considered (HCOO−···H2O and OH−, which represent different stages of nucleophile (water) activation), solvent was found to increase the reaction barriers and decrease the reaction exothermicity. Furthermore, the geometries of essential stationary points along the reaction pathway were often significantly altered. These results suggest that future studies of the hydrolysis of natural, damaged, or

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