Tautomerism of Uracil and 5-Bromouracil in a Microcosmic

Jul 11, 2007 - The base tautomerization processes of uracil/5-bromouracil were investigated in a microcosmic environment with both H2O and Na+ (W-M ...
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J. Phys. Chem. B 2007, 111, 9347-9354

9347

Tautomerism of Uracil and 5-Bromouracil in a Microcosmic Environment with Water and Metal Ions. What Roles Do Metal Ions Play? Xingbang Hu, Haoran Li,* Lei Zhang, and Shijun Han Department of Chemistry, Zhejiang UniVersity, Hangzhou 310027, People’s Republic of China ReceiVed: February 3, 2007; In Final Form: May 19, 2007

The base tautomerization processes of uracil/5-bromouracil were investigated in a microcosmic environment with both H2O and Na+ (W-M environment). It was found that uracil was more stable in the W-M environment than in the microcosmic environment with only water, which suggested that the metal ions and water work cooperated to maintain the classical nucleic acid bases. However, 5-bromouracil, a chemical mutagen, was found to be less stable than uracil in the W-M environment. Why the 5-bromouracil is easier to tautomerize and therefore induce gene mutation was explained to some extent. Further research revealed that the water molecule would assist the tautomerization in the W-M environment. However, the metal ions in different regions play absolutely opposite roles: in one region, the metal ions can prevent the base from tautomerizing, whereas in another region, the metal ion can assist the tautomerization process. Furthermore, from the viewpoint of ionization of the base, it seems BrU has a stronger tendency to lose the proton at N3, which is an intrinsic consequence of the bromine atom and is not affected by the metal cation.

Introduction Generally, the canonical nucleic acid bases (adenine, guanine, thymine, uracil, and cytosine) exist as the main forms in the double helix. The formation of specific purine-pyrimidine Watson-Crick hydrogen bonds is responsible for the maintenance of the genetic code.1 However, the enol form tautomers of the base can also be formed by proton-transfer reaction. Such tautomers may cause the nucleic acid bases to mispair, which has been proven to be one of the origins of gene mutation.2 Many noncanonical tautomeric forms of nucleic acid bases are generated by proton-transfer processes and proton transfer is a common phenomenon in the chemical3 and biological sciences.4,5 As is well-known, nucleic acids are exposed to water in most biological structures. It has also been long recognized that explicit water could accelerate the tautomerization processes from canonical base to rare base. Such a phenomenon has been called water assisting proton transfer.6,7 Water can also make the tautomerization equilibrium move toward the rare one.7,8 These researches have enhanced our perception of the roles of water in base tautomerization. As indicated in our former articles, for the tautomerization of uracil, three cooperative water molecules could decrease the barrier by more than 100 kJ/mol and decrease the Gibbs free energy by more than 20 kJ/mol.7a,b As a result, the keto form of uracil can tautomerize to its enol form at a rate of 2 s-1 and the equilibrium constant of the tautomerization reaction should be 2 × 10-6.7a,b Such rapid tautomerization and high proportion of the rare enol tautomer will lead to a quite unstable biological process and the gene mutation may happen frequently.2a,16 Hence, we shoud take more factors into consideration when we investigate gene mutation induced by base tautomerism As we know, metal ions play important roles in the biological processes. Experimental9,10 and theoretical10,11 results have * Address correspondence to this author. Fax: +86-571-8795-1895. E-mail: [email protected].

already demonstrated that metal ions can effectively bind with nucleic acid bases and affect the character of bases. These binding metal ions can influent the formation of canonical and noncanonical forms of DNA, such as triplexes, quadruplexes, bulges, and junctions.12 What roles metal ions play in the biological processes are very complicated. The bases can exist in different forms (canonical form or rare form) and the metal ions will bind with them at different sites.13 Alkali metal ion binding is expected to lead to an increase in the stability of the A-U base pairs and corresponding base when the microcosmic environment includes only metal ion.10b,c However, there is also research that indicates that explicit metal ions could stabilize the rare tautomers.13,14 Both water and metal ions are crucial for life. A microcosmic environment with both water molecules and metal ions is more appropriate for the simulation of “a real biological environment”. To investigate the taumorizing processes of the base in such an environment could disclose more essential characters of base. Uracil (U) and 5-bromouracil (BrU) were chosen for this purpose and the corresponding tautomerism of these bases will be carefully studied. 5-Bromouracil is a base analogue of uracil, which can be generated during inflammation.15 It has been widely accepted that BrU is a chemical mutagen that can mismatch with guanine (G) during DNA replication.2a,16 Though the consequence for incorporating a 5-bromouracil base on DNA/RNA mismatch is currently a focus of research, the origin of its mutagenic property is still ambiguous. There are many mechanisms explaining the mutagenicity of 5-bromouracil. One viewpoint is that the presence of the Br atom at C5 of BrU increases the proportion of the rare enol tautomer, and the enol form is the origin of the mutagenicity.2a,16a,b Another viewpoint is that BrU-G mispairing exists in a wobble model and BrU-G is more stable than the T-G mispairing.17 Some proposed that BrU is easy to change to an anionic form that pairs with G efficiently.17,18 Though there does exist some experimental support for each proposal above, direct theoretical evidence is still absent.

10.1021/jp0709454 CCC: $37.00 © 2007 American Chemical Society Published on Web 07/11/2007

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Figure 1. The tautomerization processes of U/BrU in a microcosmic environment with only H2O (W environment), calculated at B3LYP/6311++G**. The italic values represent relative enthalpies (kJ/mol). The values in parentheses belong to the isolated U/BrU isomerizations.

What roles do metal ions play in the base tautomerization? How does the water molecule cooperate with metal ions to affect the base tautomerization process? The present paper will address these questions by a systematic investigation of the tautomerism of uracil/5-bromouracil in the W-M environment. The roles played by metal ions and water molecules in the tautomerization will be discussed in detail.

the electron density becomes a minimum value along the bond path. The electron density at a BCP correlates with the strength of an atomic interaction.25 The binding enthalpy of U/BrU to the water molecule and the metal ion has been determined by

Computational Methods

E(Br)U, EM, and EW represent the enthalpy of U/BrU, metal ion, and water molecule, respectively.

The calculated results of base-metal ion systems obtained by both MP2 theory10,19 and DFT methods13,20 can well describe the geometrical parameters and energetic values. The DFT approach is also appropriate for studying base-water systems.7,8 Therefore, all of the structures were optimized with B3LYP21a and MP221b theory in the presented work. To investigate the relativistic effect of bromine, three methods were used in this research: (A) 6-311++G** was applied to all of the atoms of the molecule; (B) LANL2DZ was applied to only the Br atom and 6-311++G** was applied to the other atoms; and (C) LANL2DZ was applied to all of the atoms of the molecule. The LANL2DZ basis set includes the relativistic effect of the bromine atom. The calculated results of methods B and C are listed in the Supporting Information. Energy, frequency calculation, as well as zero-point energy (ZPE) correction have been performed at the same level of theory. The effect of basis set superposition error (BSSE) has been calculated by means of the counterpoise correction method at B3LYP/6-311++G**.22 All calculations have been performed with the Gaussian 03 suite of programs.23 The computed stationary points have been characterized as minima or transition states by diagonalizing the Hessian matrix and analyzing the vibrational normal modes. In this way, the stationary points can be classified as minima if no imaginary frequencies are shown or as transition states if only one imaginary frequency is obtained. The intrinsic reaction coordinate (IRC) was followed with the DFT method to make sure that the transition state does connect the expected reactants and products.24 It had been indicated that for ionic systems, the theoretical calculations performed in the gas phase and in the condensed phase may be a little different.5c Results obtained in many pioneering theoretical researches on nucleic acid bases with metal ions performed in the gas phase agree well with experiment,10,13 hence, we did the investigation in the gas phase likewise. The atoms in molecules (AIM)25 and natural bond orbital (NBO) analysis were performed at the B3LYP/6-311++G** in order to investigate the nature of the base-metal ion interaction. In the AIM analysis, topological properties of the electron density are analyzed to define the bond path between bonding (or interacting) atoms. Chemical bondings can be characterized by a so-called bond critical point (BCP), where

∆EBE ) E(Br)U-M-W - E(Br)U - EM - EW

Results and Discussion Because four kinds of microcosmic environments for base tautomerization were investigated in the present work, for a convenient discussion, we adopted the following abbreviations: (1) “W-M environment” represents the microcosmic environment with both Na+ and H2O; (2) “W environment” represents the microcosmic environment with only H2O; (3) “M environment” represents the microcosmic environment with only Na+; and (4) “isolated” described the situation when there is nothing in the microcosmic environment. Although uracil/5-bromouracil (U/BrU) has many tautomeric forms, those exhibiting a hydrogen atom at the N1 position (the N1-H bond is necessary for U/BrU to bind with DNA/RNA), U*/BrU* (Figure 1), are the most stable tautomers.5d,26 At the same time, in the mispairing model, the tautomer pairing with G is also U*/BrU*.2a,16 Hence we mainly focus on the isomerizations from U to U* and from BrU to BrU*. 1. Tautomerization Processes in the Microcosmic Environment with Only H2O. As shown in Figure 1, the energy changes of the tautomerizations of U-W and BrU-W are almost equal. Compared with U, there is no advantage for BrU to tautomerize to its enol forms. On the other hand, U*-W is a little more favorable than BrU*-W. Previous research has also suggested that though the influence of water on the tautomerism was examined, compared with the isomerization of U, no greater population of BrU* was predicted.5d,7c,26c What effect does bromine substitution of U at position 5 bring? Previous experiment27 and hypothesis2a,16a suggested that the introduction of the Br atom increased the proportion of the rare enol tautomer. However, the theoretical research with isolated environment or M environment presented above and elsewhere5d,7b,c,26c does not support this suggestion. In this case, it is difficult to explain the mutagenic property of 5-bromouracil. On the other hand, if the biological molecules work in a pure water environment, the corresponding rapid tautomerization rate and high proportion of the rare enol tautomer of uracil7a,b will lead to quite unstable biological processes.2a,16 2. Microcosmic Environment of Uracil/5-Bromouracil with Both Water Molecules and Metal Ions. In fact, many structural features of nucleic acids that are necessary for biological functions depend on their interactions with explicit

Tautomerism of Uracil and 5-Bromouracil

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Figure 2. Binding sites of water molecules and metal ions in the vicinity of uracil (X ) H) and bromouracil (X ) Br).

metal ions and water molecules. Previous experimental10,14 and theoretical10,13 studies have suggested that metal ions (such as Na+, K+, Mg2+, Ca2+, etc.) can bind with nucleic acid base efficiently. Additionally, the bindings between U/BrU and water molecules are also energy favorable.5d,7,26,28,29 Therefore, when we investigate the tautomerism of U and BrU, we must take the effects of both metal ions and water molecule into consideration. In the vicinity of the U/BrU (Figure 2), two different binding sides (Na1, Na2) for metal ions have been reported.10,13 In a pure water environment, 1D and 2D heteronuclear overhauser spectroscopy has revealed that there are three binding sites for water molecules (W1, W2, W3).30 Theoretical studies gave similar results.7,26,28,29 When metal ions and water molecules coexist in the environment, whether they can bind with U/BrU at these sites was still unknown. Because W2 and W3 have quite small influences on the isomerizations of U/BrU,7a we do not take W2 and W3 into consideration in the following sections. The calculated binding enthalpies between H2O and U/BrU or U-Na/BrU-Na are listed in Table 1 and these values between Na+ and U/BrU or U-W1/BrU-W1 are listed in Table 2. The values listed in Table 1 show that when there is no Na+ in the environment, the binding energy between U/BrU and water is almost the same. Single Na+ binding with U/BrU makes their binding with water less favorable and BrU-Na1 seems to be more favorable to bind with water than U-Na1. It is interesting to find that two Na+ binding with U/BrU make the binding between U/BrU and water more favorable than that of a single Na+. At the same time, the larger difference between the binding processes of U and BrU is observed, and the water molecule seems to be more favorable to bind with BrU-Na12. The values listed in Table 2 are 5-10 kJ/mol larger than those obtained at MP2(full)/6-311+G(2d,2p)//MP2(full)/6-31G* and threshold collision-induced dissociation (TCID) experiments.10b However, the systematic errors become much smaller when we calculate the energy change from U/BrU to U*/BrU*. Our calculated binding enthalpies agree well with the mass spectrometry experimental results.9b In fact, in many recent papers on the base-metal ion systems, it has been proven that the density functional theory is accurate enough to reproduce geometrical parameters, spectroscopic and energetic values with

an error close to the uncertainty in the experimental measure (about 8.0-12.0 kJ/mol).13 As indicated in Table 2, the binding energies between Na+ and O8 are 16.6 and 27.4 kJ/mol larger than those between Na+ and O7 for U and BrU, respectively. The corresponding values obtained at MP2(full)/6-311+G(2d,2p)//MP2(full)/6-31G* are 12.8 and 24.1 kJ/mol, respectively.10b An additional Na+ for U/BrU-Na+ cluster seems to be less favorable than the first Na+ binding to the isolated U/BrU. However, the large binding enthalpies indicate that two Na+ binding with U/BrU synchronously is also energy favorable. Considering the bindings between Na+ and U-W1/BrU-W1, we found that these binding processes are all energy favorable (Table 2). 3. Tautomerization Processes of U/BrU in the Microcosmic Environment with Both Na+ and H2O. 3.1. Tautomerization Processes of U-W1-Na12/BrU-W1-Na12. The base tautomerizations in the W-M environment are quite different from those in an isolated environment or a W environment. By comparing the interconverisons U/BrU-W1 f U*/BrU*-W1 and U/BrUW1-Na12 f U*/BrU*-W1-Na12, it is quite clear that the Na+ greatly affects the tautomerizations and makes the base tautomerization processes of U-W1-Na12 and BrU-W1-Na12 different from each other. The W-M environment plays absolutely opposite roles on U and BrU, respectively. It increases the barrier of uracil tautomerization by 3.3 kJ/mol whereas it decreases the barrier of 5-bromouracil tautomerization by 5.1 kJ/mol (Figure 3). The barrier of BrU-W1-Na12 f BrU*W1-Na12 is 7.9 kJ/mol lower than that of U-W1-Na12 f U*W1-Na12. This indicates that BrU is easier to tautomerize than U in the W-M environment. At the same time, compared with the tautomerizations in the W environment, two Na+ cooperating with a single water molecule reduce the stabilities of U* and BrU* by 18.8 and 8.9 kJ/mol, respectively, which indicates that the existence of Na+ stabilized the classical keto form. 3.2. Roles of Metal Ions and Water Molecule in the Tautomerization Processes. Why do metal ions make the classical base more stable and show different influences on U and BrU? To further understand the roles of metal ions in the base isomerizations and find out how a water molecule cooperates with metal ions, more tautomerization processes have been investigated (Figure 4). As we know, a negative 32F reveals excess potential energy at the BCP, which indicates the shared electron (covalent) interactions. A positive 32F at the BCP reflects an excess of kinetic energy in a bond, which indicates the closedshell (electrostatic) interactions.25a,c The 32F values listed in Tables 3 and 4 are all positive, which suggests that the Na‚‚‚O and Na‚‚‚Br interactions have electrostatic character. 3.2.1. Role of Na1+. As shown in Figure 4, when Na+ binds with O8 of U/BrU, there exist some differences between U-Na1 and BrU-Na1. The Na1+ only binds with O8 of U and the corresponding Na1‚‚‚O8 distance is 2.104 Å, which agrees well with previous results calculated at MP2(full)/6-31G* (2.109 Å)10b and B3LYP/6-311+G(2df,2p) (2.098 Å).13a However, the Na1+ binds with O8 and Br9 of BrU synchronously.

TABLE 1: The Binding Enthalpies (kJ/mol) between H2O and U/BrU or U-Na/BrU-Na at 298 Ka

a

complex

H298

complex

H298

complex

H298

U-W1 U*-W1 BrU-W1 BrU*-W1

-30.9 (-28.6) -44.0 (-39.7) -31.0 (-28.7) -44.3 (-39.6)

U-W1-Na1 U*-W1- Na1 BrU-W1- Na1 BrU*-W1- Na1

-13.7 (-9.3) -69.3 (-58.7) -22.8 (-19.1) -69.8 (-60.5)

U-W1- Na12 U*-W1- Na12 BrU-W1- Na12 BrU*-W1- Na12

-30.3 (-24.9) -62.0 (-49.5) -40.0 (-35.8) -62.6 (-49.1)

Calculated at the B3LYP/6-311++G** level of theory, including ZPE correction. Values in parentheses also include BSSE correction.

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TABLE 2: The Binding Enthalpies (kJ/mol) between Na+ and U/BrU or U-W1/BrU-W1 at 298 Ka H298

H0b

H0c

exptl

U-Na1

-152.4 (-145.0)

-151.1

-142.5 (-134.5)

U-W1-Na1f

-135.2 (-125.4)

U-Na2 BrU-Na1 BrU-Na2 U*-Na1 BrU*-Na1 U-Na12 BrU-Na12 U*-Na12 BrU*-Na12

-135.8 (-127.8) -156.4 (-150.5) -129.0 (-120.9) -69.8 (-60.1) -97.9 (-92.2) -79.3 (-74.3) -76.3 (-72.3) -42.0 (-34.8) -58.1 (-54.1)

-134.7 -155.1 -128.0

-129.7 (-122.8) -147.2 (-138.0) -123.1 (-116.2)

-141.0 ( 4.0d -134.6 ( 3.4e -142.3 ( 4.8e

BrU-W1-Na1

-187.2 (-179.1)

U*-W1-Na1 BrU*-W1-Na1 U-W1-Na12 BrU-W1-Na12 U*-W1-Na12 BrU*-W1-Na12

-95.1 (-82.1) -148.8 (-138.9) -78.7 (-70.8) -124.3 (-117.9) -59.9 (-46.1) -101.8 (-89.0)

complex

complex

H298

a Calculated at the B3LYP/6-311++G** level of theory, including ZPE correction. Values in parentheses also include BSSE correction. b Calculated at 0 K. c Calculated at 0 K at MP2(full)/6-311+G(2d,2p)//MP2(full)/6-31G* level of theory.10b d Tandem mass spectrometry experiment result.9b e Threshold collision-induced dissociation experiment result.10b f Very small imaginary frequency (about -6.0 cm-1) was found in the evaluation of ZPE.

Figure 3. The tautomerization processes of U/BrU in a microcosmic environment with both H2O and Na+ (W-M environment), calculated at B3LYP/6-311++G**.

Figure 4. The tautomerization processes of U/BrU in a microcosmic environment with only Na+ (M environment) and in a microcosmic environment with single Na+ and H2O, calculated at B3LYP/6-311++G**.

The corresponding Na1‚‚‚O8 distance is 2.165 Å, which is shorter than previous results calculated at MP2(full)/6-31G* (2.199 Å).10b

The Na1+ can increase the barrier of base tautomerization and stabilize the classical keto form (stabilization role). Furthermore, it will make the base tautomerizations of U and BrU

Tautomerism of Uracil and 5-Bromouracil

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TABLE 3: Bond Critical Point (BCP) Properties of Na1‚‚‚O8 and Na1‚‚‚Br9 Interactions complex

FNa1‚‚‚O8a

32FNa1‚‚‚O8b

U-Na1 ts-U-Na1 U*-Na1 BrU-Na1 ts-BrU-Na1 BrU*-Na1 U-W-Na1 ts-U-W-Na1 U*-W-Na1 BrU-W-Na1 ts-BrU-W-Na1 BrU*-W-Na1

0.0316 0.0288 0.0253 0.0296 0.0268 0.0220 0.0309 0.0300 0.0277 0.0289 0.0275 0.0246

0.2473 0.2083 0.1696 0.2098 0.1817 0.1447 0.2294 0.2094 0.1899 0.1998 0.1856 0.1644

FNa1‚‚‚Br9a

32FNa1‚‚‚Br9b

0.0078 0.0073 0.0130

0.0330 0.0305 0.0589

0.0100 0.0127 0.0128

0.0442 0.0581 0.0583

a The electron density at the BCP. b The Laplacian of the electron density at the BCP.

different from each other. Comparing the interconverisons U/BrU f U*/BrU* (Figure 1) and U/BrU-Na1 f U*/BrU*Na1 (Figure 4), it is quite clear that the Na1+ increase the barrier of proton transfer by 30.9 and 27.1 kJ/mol for U and BrU, respectively. When Na1+ binds with U/BrU, compared with isolated U/BrU, the proton must transfer a longer distance to get to the transition states (Figure S1 in the Supporting Information), thus, the base tautomerization with Na1+ becomes harder correspondingly. On the other hand, Na1+ reduces the stability of both U* and BrU* by 82.5 and 58.4 kJ/mol, respectively, which indicates that the Na1+ greatly stabilizes the classical keto form. At the same time, BrU*-Na1 becomes more stable than U*-Na1 (Figure 4). As listed in Tables 3 and 4, the electron density between Na1 and O8 (FNa‚‚‚O8) in the base interconverison of U-Na1 f U*-Na1 decreases from reactant to product. The weakened Na1‚‚‚O8 interaction will make the tautomerization process quite unfavorable. However, for BrU-Na1 f BrU*-Na1, though the FNa1‚‚‚O8 value decreases from reactant to product, the FNa1‚‚‚Br9 value increases concomitantly. The strengthening of Na1‚‚‚Br9 interaction could compensate for the instability induced by the weakening of the Na1‚‚‚O8 interaction. As a result, BrU*-Na1 is more stable than U*-Na1. By comparing the tautomerizations U/BrU-W1 f U*/BrU*W1 and U/BrU-W1-Na1 f U*/BrU*-W1-Na1, it is also found that Na1+ can increase the barrier of base tautomerization and stabilize the classical keto form. Furthermore, it will make the base isomerizations of U and BrU different from each other. The Na1+ obviously affects the interaction between U/BrU and the water molecule. The Na1+ weakens the OW-H‚‚‚O8

hydrogen bond in U-W-Na1 and BrU-W-Na1 (Table 5). However, it remarkably enhances the O8-H‚‚‚OW hydrogen bond in U*-W-Na1 and BrU*-W-Na1. Then, the water molecule in U*W/BrU*-W stabilizes U*/BrU* by 13.2 and 13.4 kJ/mol, respectively (Figure 1), whereas it stabilizes U*-Na1/BrU*-Na1 by 55.6 and 46.0 kJ/mol, respectively (Figure 4). 3.2.2. Role of Na2+. As shown in Figure 4, the interactions between Na2+‚‚‚U and Na2+‚‚‚BrU are quite similar. Na2+ only binds with O7 in the keto form and binds with O7 and N3 in the enol form. The corresponding Na2‚‚‚O7 and Na2‚‚‚N3 distances in U*-Na2 are 2.213 and 2.572 Å, which are close to previous results calculated at B3LYP/6-311+G(2df,2p) (2.203 and 2.563 Å, respectively).13a The Na2+ assists the base tautomerization process of both U and BrU (mutagenicity role). From the comparison of U/BrU f U*/BrU* (Figure 1) and U/BrU-Na2 f U*/BrU*-Na2 (Figure 4) processes, it is clear that the Na2+ lowers the barrier of proton transfer by 21.6 kJ/mol for both U and BrU. When Na2+ binds with U/BrU, compared with isolated U/BrU, the proton could move only a shorter distance to get to the transition states (Figure S1 in the Supporting Information), as a result, the base tautomerization with Na2+ becomes more favorable. On the other hand, Na2+ increases the stability of U* and BrU* by 51.1 and 47.2 kJ/mol, respectively, which indicates that the Na2+ greatly stabilizes the nonclassical enol form. Comparing the interconverisons U/BrU-Na1 f U*/BrU*-Na1 and U/BrU-Na12 f U*/BrU*-Na12, it is also found that Na2+ can reduce the barrier of base tautomerization by 21.3 and 19.9 kJ/mol for U and BrU, respectively. At the same time, Na2+ increases the stability of U*-Na1 and BrU*-Na1 by 45.2 and 40.2 kJ/mol, respectively. 3.2.3. Role of H2O and the Cooperation between H2O and Na+. Previous research has disclosed that water can assist many base interconverisons.6,7 Similar functions of water molecules were also observed in the tautomerization in the W-M environment. Is the role played by the water molecule in the W environment the same to that in the W-M environment? We are quite interested in the cooperative effect between H2O and Na+. The FNa1‚‚‚O8 value of U-W-Na1 is smaller than that of U-Na1, whereas the FNa1‚‚‚O8 values of ts-U-W-Na1 and U*-W-Na1 are larger than those of ts-U-Na1 and U*-Na1. It shows that the water molecule weakens the Na1‚‚‚O8 interaction in the reactant, whereas it enhances the Na1‚‚‚O8 interactions in the transition state and the product. The same results can be deduced by comparing the BrU-Na1 f BrU*-Na1 and BrU-W1-Na1 f BrU*-W1-Na1 processes (Table 3). As a result, by comparing the tautomerization processes U/BrU-Na1 f U*/BrU*-Na1 and

TABLE 4: Bond Critical Point (BCP) Properties of Na1‚‚‚O8, Na2‚‚‚O7, and Na1‚‚‚Br9 Interactions

a

complex

FNa1‚‚‚O8a

32FNa1‚‚‚O8b

FNa2‚‚‚O7a

32FNa2‚‚‚O7b

U-Na12 ts-U-Na12 U*-Na12 BrU-Na12 ts-BrU-Na12 BrU*-Na12 U-W-Na12 ts-U-W-Na12 U*-W-Na12 BrU-W-Na12 ts-BrU-W-Na12 BrU*-W-Na12

0.0248 0.0227 0.0175 0.0249 0.0221 0.0163 0.0254 0.0249 0.0226 0.0238 0.0232 0.0210

0.1847 0.1543 0.1077 0.1734 0.1449 0.1001 0.1766 0.1660 0.1475 0.1556 0.1506 0.1358

0.0258 0.0261 0.0277 0.0243 0.0256 0.0269 0.0257 0.0280 0.0277 0.0252 0.0275 0.0273

0.1909 0.1962 0.2080 0.1800 0.1906 0.2010 0.1928 0.2129 0.2103 0.1883 0.2092 0.2068

The electron density at the BCP. b The Laplacian of the electron density at the BCP.

FNa1‚‚‚Br9a

32FNa1‚‚‚Br9b

0.0000 0.0000 0.0108

0.0000 0.0000 0.0478

0.0084 0.0113 0.0111

0.0365 0.0511 0.0498

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TABLE 5: The Influence of Na+ on the Interaction between U/BrU and Water Moleculea donor

acceptor

∆E2 (kJ/mol)

LP(1) O 8 LP(2) O 8

between U(U-Na1) and W BD*(1) OW-H BD*(1) OW-H

6.6 (2.5) 24.5 (17.1)

LP(2) OW

between U*(U*-Na1) and W BD*(1) O8-H

86.1 (157.4)

LP(1) O 8 LP(2) O 8

between BrU(BrU-Na1) and W BD*(1) OW-H 5.4 (2.3) BD*(1) OW-H 19.3 (13.3)

LP(2) OW

between BrU*(BrU*-Na1) and W BD*(1) O8-H 94.1 (156.5)

a Natural bond orbital (NBO) analysis results. LP denotes the occupied lone pair and BD* denotes the formally empty antibonding orbital. Values in parentheses belong to the U-W-Na1 and BrUW-Na1 systems. Values out of parentheses belong to the U-W and BrU-W systems.

U/BrU-W1-Na1 f U*/BrU*-W1-Na1 (Figure 4), it is clear that the additional water molecule lowers the barrier of proton transfer by 120.3 and 129.6 kJ/mol for U and BrU, respectively. By comparing the tautomerizations U/BrU f U*/BrU* and U/BrU-W1 f U*/BrU*-W1, it is clear that the water molecule lowers the barrier of proton transfer by 109.6 and 111.2 kJ/mol for U and BrU, respectively. It suggests that Na+ could cooperate with water and affect the base isomerization synchronously. There are two important differences between the interconverisons in the W environment and the W-M environment, which suggest that there exists a cooperative effect between H2O and Na+. First, the assisting ability of the water molecule in the W environment is weaker than that in the W-M environment. Second, the barrier of ts-U-W1 is almost the same with that of ts-BrU-W1, and so is the barrier of ts-U-Na1 and ts-BrU-Na1. However, the cooperative effect between Na+ and single water makes the barrier of ts-BrU-W1-Na1 lower than that of ts-U-W1-Na1. When two Na+ bind with O8 and O7, respectively, using the same method mentioned above, we found that two Na+ can also cooperate with the water molecule to affect the base isomerizations. Other evidence of the cooperation between Na+ and H2O comes from the variations of geometric configuration (Figure 4) and AIM analysis results (Tables 3 and 4). In the BrU interconverison, the Na1‚‚‚Br9 bond length is increased by 0.025 and 0.063 Å in BrU-Na1 f ts-BrU-Na1 and BrU-Na12 f ts-BrU-Na12 and decreased by 0.263 and 0.506 Å in ts-BrUNa1 f BrU*-Na1 and ts-BrU-Na12 f BrU*-Na12. It is worth noting that the variation of the Na1‚‚‚Br9 bond from TS to product is larger than that from reactant to TS when there is no explicit water molecule. However, when there is an explicit water molecule, the variation of the Na1‚‚‚Br9 bond from TS to product is smaller than that from reactant to TS. The Na1‚‚‚Br9 bond length is decreased by 0.251 and 0.137 Å in BrU-W1Na1 f ts-BrU-W1-Na1 and BrU-W1-Na12 f ts-BrU-W1-Na12

and changed by 0.001 and 0.010 Å in ts-BrU-W1-Na1 f BrU*W1-Na1 and ts-BrU-W1-Na12 f BrU*-W1-Na12. The AIM analysis also shows that the Na1‚‚‚Br9 interaction is enhanced more in the conversion from TS to product when there is no explicit water molecule; however, this interaction can be enhanced more in the conversion from reactant to TS when there is an explicit water molecule. 4. Influence of Metal Ion on the Ionization of the Bases. Previous research on the tautomerism of U/BrU performed in the gas phase and water solution indicated that there were no relevant differences in the intrinsic tautomeric preference of these bases. On the basis of this, it was suggested that the mutagenic properties of 5-bromouridine should stem from its ability to lose the proton at N3.26c We further examined the influence of metal ion on the ionization of uracil and its 5-bromo derivative. The calculated N3-H deprotonation enthalpy (DPE) of uracil is 1437.1 kJ/mol, which is 11.0 kJ/mol lower than that value calculated at B3LYP/6-31+G(d,p).26a As shown in Table 6, the Na+ can reduce the DPE values of both U and BrU by 349.0 and 343.5 kJ/mol, respectively. Taking the water in the microcosmic environment into consideration, the Na+ can also reduce the DPE values of both U and BrU by 366.1 and 351.5 kJ/mol, respectively. This indicates that the explicit metal ion can facilitate the ionization of U and BrU. It is worth noting that whether there is explicit metal ion or not, BrU seems to have a stronger tendency to lose the proton at N3, which demonstrates that the ionization preference of BrU is an intrinsic consequence of the bromine atom. About the origin of the mutagenicity of BrU, enolization and ionization are two dominating proposals. Until now, there was no model that could explain the mutagenicity of BrU based on both enolization and ionization.16-18 Our model including the metal ion shows that both the enolization and the ionization of BrU are more favorable than those of U. 5. Results Including the Relativistic Effects. The atomic number of bromine is 35. Though the parameters for bromine are available in the 6-311++G** basis set employed in this work, to make the results more credible, it is necessary to consider the relativistic effect of bromine. Two additional methods were tested: methods B and C as described in the computational method section. Actually, method B (LANL2DZ was applied to only the Br atom and 6-311++G** was applied to the other atoms) was often adopted in previous works and generated satisfying results compared with experiment, whereas method C (LANL2DZ was applied to all of the atoms of the molecule) was seldom adopted.10b,31 The results obtained by method B agree very well with these obtained at B3LYP/6-311++G** (Table 7). The binding enthalpy between BrU and Na1 obtained by method B is 149.9 kJ/mol (including ZPE and BSSE correction), which is quite close to the experiment value (142.3 ( 4.8 kJ/mol).10b The values obtained by method C are larger than those obtained at B3LYP/6-311++G** (Table S2 and Section 2 in the Supporting Information); however, it can still support our standpoint about the base tautomerization of U/BrU proposed in this paper.

TABLE 6: The Deprotonation Enthalpies (DPE, in kJ/mol) of N3-H of U and BrU DPE Uf +H U-Na1+ f U-Na1 + H+ U-W f U- + H3O+ U-W-Na1+ f U-Na1 + H3O+ U-

+

1437.1 1088.1 797.1 431.0

DPE BrU f +H BrU-Na1+ f BrU-Na1 + H+ BrU-W f BrU- + H3O+ BrU-W-Na1+ f BrU-Na1 + H3O+ BrU-

+

1403.0 1059.5 763.1 411.6

Tautomerism of Uracil and 5-Bromouracil

J. Phys. Chem. B, Vol. 111, No. 31, 2007 9353

TABLE 7: Relative Enthalpies (kJ/mol) Including the Relativistic Effectsa BrU f BrU* BrU-W1 f BrU*-W1 BrU-Na1 f BrU*-Na1 BrU-W1-Na1 f BrU*-W1-Na1 BrU-Na2 f BrU*-Na2 BrU-Na12 f BrU*-Na12 BrU-W1-Na12 f BrU*-W1-Na12

∆qHb

∆H

175.4 64.3 203.2 73.7 153.7 183.2 61.5

52.1 38.4 111.7 64.6 6.4 71.7 49.4

a LANL2DZ was applied to Br atom and 6-311++G** was applied to other atoms. b The barrier of the transition state.

Conclusions The tautomerization processes of uracil/5-bromouracil (U/ BrU) have been systematically investigated in a model including U/BrU, water molecule, and metal ions. In an effort to unveil the roles played by metal ions in the base isomerization and the cooperative effect between the water molecule and metal ions, tautomerizations with only a single Na+, with only two Na+, with a single Na+ and H2O were investigated. On the basis of the obtained results, the following can be safely stated: (1) It is more difficult for U to tautomerize in the microcosmic environment with both Na+ and H2O (W-M environment) than in the microcosmic environment with only H2O (W environment). That is to say, the W-M environment is very important for U to maintain the classical nucleic acid base form and avoid gene mutation. (2) Compared with U, BrU is easier to tautomerize and BrU* is more stable than U* in the W-M environment, which suggests a higher mutagenicity of BrU. (3) The Na1+ can increase the barrier height of the base tautomerization and stabilize the classical keto form. Furthermore, Na1+ can make the base tautomerization processes of U and BrU different from each other. (4) The Na2+ can assist the base isomerization. Its effect for the base tautomerizations of U and BrU is the same. (5) There is a cooperative effect between Na+ and H2O, which makes the tautomerization processes in the W-M environment quite different from those in the W environment and the M environment. (6) From the viewpoint of ionization of the base, it seems BrU has a stronger tendency to lose the proton at N3, which is an intrinsic consequence of the bromine atom and is not affected by the metal cation. The origin of the mutagenicity of nucleic acid base is of great interest in research, but still remains a controversial topic due to the gene mutation processes being quite complicated. It is not possible to solve this problem only by experiment or by theoretical calculation, because each method can solve part of the problem. Our understanding of the structural tautomer interconversion of uracil/5-bromouracil has improved greatly via investigation of the base tautomerization with not only water but also metal ions, both of which are important components for the real biological environment. We expect it will be helpful for the future development of research on the tautomerism of uracil/5-bromouracil. Acknowledgment. This work was supported by the National Natural Science Foundation of China (Nos. 20573093 and 20434020). Supporting Information Available: The relation between the barrier of the transition state and the proton-transfer distance, the tautomerization of U-Na2, results obtained at MP2/

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