Primary Steps in the Reaction of OH Radicals with Peptide Systems

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Primary Steps in the Reaction of OH Radicals with Peptide Systems: Perspective from a Study of Model Amides Hoang Q. Doan, Alexander C. Davis, and Joseph S. Francisco* Department of Chemistry, Purdue UniVersity, West Lafayette, Indiana 47907-2084 ReceiVed: January 14, 2010; ReVised Manuscript ReceiVed: March 9, 2010

This study reports on an ab initio investigation into the effects of radical attack on peptide backbones using eight model amides. The eight model amides are divided into two subcategories, formamide and acetamide. Within each subcategory, there are four unique systems which include the parent, cis and trans methyl, and the dimethyl structures. The mechanism for hydrogen abstraction shows R-C abstraction is kinetically and thermodynamically favorable for all formamide systems. The addition of a methyl group on the nitrogen results in a secondary competitive pathway at the γ-C site. In the parent acetamide, β-abstraction is preferred. The addition of a methyl group on the amino group of acetamide results in the introduction of a secondary pathway that will outcompete β-abstraction, both thermodynamically and kinetically. For all systems, H-abstraction off the nitrogen is the least preferred path. With the addition of a methyl group on the nitrogen, H-abstraction off the nitrogen becomes more favorable. To explain site preference, geometry structures and stabilizations are examined. The results of this study have implications for future studies on radical attack of peptide and protein systems. Fe2 + H2O2 f Fe3 + OH- + •OH

I. Introduction Reactive oxygen species (ROS) have been implicated in numerous physiological pathologies such as cancer, atherosclerosis, and Alzheimer’s disease.1-4 ROS are generated in biological systems endogenously (e.g., as natural byproducts of cellular metabolism) and exogenously (e.g., metal-ioncatalyzed reactions and ionizing radiation).1,3,5,6 They include both radical (e.g., •OH, •O2-, and ROO•) and nonradical species (e.g., H2O2, HOCl, CO, and O3).4,7,8 In limited concentrations, ROS are beneficial to certain biological systems such as signaling pathways.8 Normally, endogenous enzymes and antioxidant defense mechanisms can remediate excess ROS.4,6 However, under certain circumstances, elevated concentrations can lead to a state of oxidative stress (OS) which can result in irreparable destruction of cell function.4,5 Free radicals are a subclass of ROS which contain one or more unpaired electrons.6 As a result of these unpaired electrons, free radicals are highly reactive and relatively unstable.6,9 The hydroxyl radical (•OH) has a half-life in solution of less than 10-9 s.6 Their presence in biological systems, though short-lived, can cause irreversible damage to important biological macromolecules including lipids, nucleic acids, carbohydrates, and proteins.5 The •OH can be produced through inflammatory processes, OS in biological systems, and the reaction between • O2- and H2O2 in the Haber-Weiss or Fenton reaction using copper/iron and H2O2.1,5,6,10,11 Haber-Weiss: •

O2- + H2O2 f •OH + OH- + O2

Fenton: * To whom correspondence should be addressed.

Proteins are major targets for ROS attack due to their high concentrations in biological systems.5 Several papers have reviewed the modifications and deactivations of proteins by ROS.5,9,12,13 Free radical attack can result in protein fragmentation and denaturation, which in turn alters the intramolecular forces leading to protein unfolding and possible formation of a new tertiary structure.5,12,13 Davies found •OH treated proteins readily degrade in the presence of enzymes such as proteases and peptidases, suggesting increased proteolytic susceptibility with hydroxyl attack.12 Another study investigating β-glucosidase enzyme showed that •OH can cause damage that leads to activity loss under irradiation conditions.14 Stefani and Dobson determined misfolded proteins to be a common link in a number of amyloidal diseases such as type II diabetes, rheumatoid arthritis, spongiform encephalopathy, and degenerative neurological diseases like Parkinson’s, Alzheimer’s, and Huntington’s disease.15 When proteins misfold, aggregate, and accumulate in the body, their functions change and cell death can result.16,17 Proteins can aggregate in the joints, skeletal tissues, and organs, causing hemodialysis-related and systemic amyloidosis.15 In the brain this aggregation results in neurodegenerative pathologies. Some studies point to ROS attack as the molecular mechanism behind the misfolding of proteins and aggregation, specifically for misfolded proteins in Alzheimer’s disease and the mutated Parkin protein in Parkinson’s disease.2,16 These implications indicate the importance of studying oxidative and, more specifically, radical attack on proteins. Experimental studies on the effects of radical attack on protein systems have been extensive. Techniques such as radiolysis, enzymatic hydrolysis, direct rapid-flow electron paramagnetic resonance, and other spectroscopic studies have been used to determine the selectivity of radical attack at different sites in amino acids and peptides.1,5,9,18,19 Hawkins and Davies demonstrated that less selective •OH attack occurs more rapidly at the R-carbon site than at the N-site in small peptides.18 Galano et

10.1021/jp100375c  2010 American Chemical Society Published on Web 04/06/2010

Reaction of OH Radicals with Peptide Systems

J. Phys. Chem. A, Vol. 114, No. 16, 2010 5343 only ROS found to be capable of directly cleaving proteins.9 Model amide systems are employed to mimic the peptide bond in a polypeptide chain because, given the size of proteins, a study of • OH attack on even the simplest protein is too computationally demanding for an ab initio approach. Potential energy surface diagrams, obtained from ab initio calculations, allow the comparison of kinetic and thermodynamic data of the different pathways of attack enabling the determination of the preferred site for •OH attack on the amide systems. These results can then be applied to larger peptide systems and proteins for continued computational research on protein misfolding. Such studies may be able to determine the mechanisms of protein folding diseases due to the damaging effects of oxidative stress. II. Computational Methods

Figure 1. Labeling scheme for model amides.

al. came to the same conclusion with L-alanine and glycine.20 However, Stefanic et al. and Liessmann et al. concluded that the nitrogen and R-C sites are cocompetitive for H-abstraction via radical attack.19,21 Few computational studies on free radicals and proteins have been conducted. Huang and Rauk investigated peroxy radical attack on glycine and alanine peptides along with two model amide systems using the density functional theory (DFT), B3LYP/6-31G(d) method.3 Another study by Armstrong, Yu, and Rauk applied ab initio methods to determine C-H bond dissociation energies of glycine with various radicals. They determined that the R-C is the most vulnerable site to attack.22 Lu, Li, and Lin performed DFT calculations to determine site specificity when comparing three-dimensional structure motifs in protein oxidation by •OH.10 Although performed at higher computational costs, the ab initio approach models the energetics in a radical system better than DFT.23 According to Wodrich et al. and Schreiner et al., B3LYP/6-31G(d) poorly estimates intermolecular forces resulting in increased errors for larger systems.24,25 DFT calculations contain a self-interaction error for radical systems that ab initio methods lack.26 In this study eight amide systems (Figure 1) are used to model kinetic and thermodynamic properties of •OH attack on proteins: (1) formamide; (2) trans-N-methylformamide (tNMF); (3) cisN-methylformamide (cNMF); (4) N,N-dimethylformamide (DMF); (5) acetamide; (6) cis-N-methylacetamide (cNMA); (7) transN-methylacetamide (tNMA); and (8) N,N-dimethylacetamide (DMA). These systems have an amino group bounded to a carbonyl group which makes them ideal systems for modeling the peptide linkage in polypeptide chains.27 They are often used as models in studies of solvation and spectroscopy. Patel and Brooks used ab initio methods to determine electrostatic parameters of solvated N-methylacetamide.28 These parameters reflect the desired chemical environments that future research can be based on. Other studies using model amides found them to be appropriate for studying preferential solvation in water and alcohol solutions due to their mimicry of a protein.29-32 Several papers have also investigated the microwave spectra of formamide, acetamide, and NMA with the intention of using the results to study peptide systems.33-37 This study concentrates on an ab initio approach to determining the preferred site of •OH attack on model amide systems. The •OH was selected for its abundance in biological systems, highly reactive nature, and its role as the primary radical in the modification and degradation of proteins.12,18 When coupled with oxygen, it is the

A series of ab initio calculations are performed using the Gaussian 03 suites of programs.38 Reactant, prereactive complex, transition state, and product geometries are optimized with unrestricted second-order Møller-Plesset perturbation theory (UMP2) using 6-31G(d) and 6-311G(2d,2p) basis sets. Coupled cluster single-point energy calculations that incorporate electron correlation are computed at the CCSD(T) level of theory using 6-311G(2d,2p) and 6-311++G(2df,2p) basis sets using the MP2/ 6-311G(2d, 2p) geometries. Zero point energy corrections (ZPE) are determined at the UMP2/6-31(d) and UMP2/6-311G(2d,2p). These values are applied to the UMP2 and CCSD(T) energies to correct the enthalpies of reaction and activation energy. Geometry optimizations are also performed using the CBS-Q and G2 composite methods. Transition states are confirmed by the presence of a single negative frequency which corresponds to the reaction coordinate of the abstracted hydrogen. Four species with similar chemical connectivities to the amide systems serve to evaluate the reliability of the computational methods used (Table 1). Formaldehyde and acetaldehyde model the hydrogen abstraction off R-C in the formamide species. Acetaldehyde and acetone model the H-abstraction off a methyl attached to a carbonyl group, the β-C in the acetamide species. Methylamine models two sites for radical attack that are common to all eight amides: the nitrogen and a methyl attached to the amino group, the γ-C. In addition to having similar chemical structures as the amides, these four species are commonly used in experimental studies. There are numerous kinetic results for the reaction between the •OH and the four test species.39-47 The enthalpies of formation, at 0 K, for all the test case reactants and radicals are obtained from ref 48. The experimental results are compared to the reaction enthalpies determined at the six levels of theory used in this study, to assess the reliability of each computational method. III. Results Table 1 compares the computationally and empirically determined enthalpy of reaction values at the six levels of theory for the four test species. Formaldehyde and acetone have one reaction pathway for •OH attack while acetaldehyde and methylamine have two unique sites for H-abstraction. Comparing the test cases to empirical data (Table 2), the CBS-Q level of theory shows the best agreement with experimental data, followed by G2 and CCSD(T)/6-311++G(2df,2p)//MP2/6311G(2d,2p) with root mean square values of 1.1, 1.2, and 2.5 kcal mol-1, respectively. The structures list geometries optimized at MP2/6-311G(2d,2p), which is more accurate than MP2(FC)/ 6-31G(d) used in the CBS-Q composite method (Figures 2-9).49 The CBS-Q data set is used in the potential energy surface diagrams (Figure 10).The relative energies for H-abstraction by

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TABLE 1: Calibration of Energetic for Test Cases with Zero-point Corrections and CRC Experimental Values at 0 K (kcal mol-1)47

TABLE 2: Root Mean Square (rms) Error for Calibration Cases (kcal mol-1) at 0 K

•

level of theory

rms error

CCSD(T)/6-311++G(2df,2p)//UMP2/6-311G(2d,2p) G2 CBS-Q

2.5 1.2 1.1

OH for the eight amide systems considered in this work are summarized in Table 3. The eight amides model the peptide bond in a polypeptide chain. There are three R-group sites attached to the backbone structure (Figure 1). The model set is divided into two groups: a formamide group with hydrogen at the R1 site and an acetamide group with a methyl group at this location. Within each group is a series of four model systems. The presence of hydrogen at both R2 and R3 yields the parent model system, formamide or acetamide. A single methyl group on the R2 or R3 site results in a cis-N-methyl or trans-N-methyl system, respectively. The trans and cis nomenclature used are employed because the energetics of the transition states differ in relation to the constituents of the R2 and R3 sites. N,N-Dimethyl systems have a methyl group at both the R2 and R3 sites. Each amide system has three unique reactive sites where a hydrogen atom can be abstracted. In the formamide set, these sites are on the R-C, γ-C, and nitrogen. The acetamides have hydrogen abstracted from the β-C and γ-C and nitrogen. H-abstraction at the R1 site in the formamide group set is defined as R-C H-abstraction. In the acetamides, the R1 hydrogen atom is labeled the β-C. When a methyl group is present at the R2

and/or R3, the methyl carbon is considered the γ-C atom in either case. A hydrogen at these sites gives nitrogen H-abstraction. Table 3 displays the six levels of theory with relative binding energies, activation energies, and enthalpies of reaction determined by each theory. The CCSD(T)/6-311++G(2df,2p)// UMP2/6-311G(2d,2p) and CBS-Q levels of theory are in the best agreement with each other for all prereactive complexes. G2 has consistently the highest energy, deviating by 2.0 kcal mol-1 from CBS-Q and 2.3 kcal mol-1 from CCSD(T)/6311++G(2df,2p)//UMP2/6-311G(2d,2p), while there is only a 0.3 kcal mol-1 standard deviation between CCSD(T)/6311++G(2df,2p)//UMP2/6-311G(2d,2p) and CBS-Q. For the activation energies and reaction enthalpies for all model systems, the CBS-Q shows the best agreement with G2. The average difference between the activation energies is 0.4 kcal mol-1 with a standard deviation of 0.6 kcal mol-1. For reaction enthalpies, there is only a 0.1 kcal mol-1 average difference with a standard deviation of 0.7 kcal mol-1 between CBS-Q and G2. CCSD(T)/ 6-311++G(2df,2p)//UMP2/6-311G(2d,2p) has a consistently higher barrier by approximately 3.0 kcal mol-1 and a higher enthalpy of reaction with an average difference of 1.2 kcal mol-1. A. Formamide Trends. The simplest system reported here is formamide (FM). Figure 2 shows the minimum energy structures comprising the reactant, prereactive complex, transition state, and product structures. The energetics of the potential energy surface is depicted in Figure 10a. All H-abstraction pathways for the amides contain a prereactive complex as a


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Figure 2. Reactant, prereactive complex, transition state, and product geometries for formamide. Parameters are optimized at the MP2/6-311G(2d,2p) level of theory. Bond lengths are in angstroms and bond angles are in degrees.

result of hydrogen bonding between the •OH and the functional groups on the amide. R3 abstraction has the highest binding energy while R1 and R2 abstraction have similar lower energies. The stabilization by H-bonding explains the trends in binding energies. R3 abstraction has a longer H-bond of 2.124 Å while the prereactive complex for R2 abstraction has two H-bonds with distances of 1.901 and 2.275 Å and R1 abstraction results in the shortest H-bond, 1.868 Å. With the lowest activation barrier and enthalpy of formation, R1 (R-C) abstraction is the most kinetically and thermodynamically favorable of the three pathways. R1 abstraction has an activation barrier that is approximately 9.5 kcal mol-1 lower than R2 and R3 abstraction. The N-C bond lengths in the transition state structures indicate the relative energies seen in the potential energy surface diagram. TS 1a has the shortest N-C bond, 1.347 Å, compared to 1.380 and 1.413 Å for TS 1b and TS 1c, respectively. This indicates that as the N-C bond length shortens, the activation energy decreases.

The trends in thermodynamic data are similar to the kinetic data mentioned above. R-C abstraction is a much more competitive pathway for radical attack than nitrogen H-abstraction. The enthalpy of reaction for R1 abstraction is lower than R2 and R3 abstraction by 21.4 kcal mol-1. Both the N-C and C-O bond lengths shorten for R-C H-abstraction, suggesting increased stabilization. The changes in N-C and C-O bond lengths from the reactant to product geometry for R1 abstraction are 1.358-1.344 Å and 1.214-1.196 Å, respectively. R2 and R3 H-abstractions yield the same enthalpy of reaction with an inconsistent stabilization and destabilization of bond lengths and bond angles. 1. trans- and cis-N-Methylformamide. trans-N-methylformamide (tNMF) and cis-N-methylformamide (cNMF) follow a similar trend to FM. Figures 3 and 4 depict the structures for the tNMF and cNMF systems, respectively. Parts b and c of Figure 10 summarize the H-abstraction pathways for tNMF and cNMF, respectively. Consistent with FM, the binding energy

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Figure 3. Reactant, prereactive complex, transition state, and product geometries for trans-N-methylformamide. Parameters are optimized at the MP2/6-311G(2d,2p) level of theory. Bond lengths are in angstroms and bond angles are in degrees.

for R3 H-abstraction is the least favorable for both systems. For tNMF, the R2 H-abstraction has the lowest binding energy due to two H-bonds forming. In cNMF, the methyl group at the R2 site prevents formation of a secondary H-bond. As a result, the binding energies between R1 and R2 abstraction are closer and higher in energy in cNMF than in tNMF prereactives. Also, consistent with the trends in FM, abstraction off the R1 site has the lowest activation energy in both tNMF and cNMF. H-abstraction off the nitrogen is kinetically the least favorable, while R-C and γ-C are similar for both systems. However, γ-C H-abstraction is closer to R-C in the cNMF system. In tNMF, the difference between γ-C and R-C Habstraction is 2.9 kcal mol-1 but is only 1.5 kcal mol-1 in cNMF. Examination of the TS geometries indicates that R-C and γ-C H-abstraction gives a stabilized, shorter N-C bond length.

The difference in the enthalpy of formation for R-C and γ-C H-abstraction are within the root mean square error shown in Table 2 and can therefore be considered competitive pathways. Nitrogen H-abstraction continues to be least thermodynamically favorable with an enthalpy of formation that is approximately 15 kcal mol-1 higher than R-C and γ-C H-abstraction. When product geometries are examined, γ-C H-abstraction results in a shorter methyl-nitrogen bond length. This shortened distance is associated with the delocalization of the radical site between the N-C(O) bonds. The dihedral angle for a remaining hydrogen on the radical methyl is 10.0° with a bond angle of approximately 120° reflecting a trigonal planar geometry, indicating that the radical site is parallel to the π cloud, allowing for the delocalization of the radical site which explains the lowered energy in γ-C abstraction. This was not observed in FM, because



Figure 4. Reactant, prereactive complex, transition state, and product geometries for cis-N-methylformamide. Parameters are optimized at the MP2/6-311G(2d,2p) level of theory. Bond lengths are in angstroms and bond angles are in degrees.

no methyl group was present for γ-C abstraction. These two model systems reflect the competition of radical attack between the R-C and γ-C abstraction, especially thermodynamically, and reinforces the observed unfavorability of nitrogen H-abstraction already seen in the formamide system. 2. N,N-Dimethylformamide. The energy diagram in Figure 10d of N,N-dimethylformamide (DMF) further illustrates the competition between R-C and γ-C H-abstraction observed in tNMF and cNMF. Figure 5 depicts the structures of the DMF system. Again, R3 H-abstraction has the highest binding energy, while R1 and R2 are energetically similar to each other and more favorable than R3. R1 and R2 H-abstractions are cocompetitive with a difference in binding energies within the root mean square error. The three abstraction pathways in DMF have close activation energies compared to the other formamide systems. R1 H-

abstraction is the most kinetically favorable with a 1.5 kcal mol-1 difference from R2. R3 H-abstraction is the least kinetically favorable, with an activation energy that is 2.6 and 1.1 kcal mol-1 higher than R1 and R2 H-abstraction, respectively. This trend correlates to the N-C bond lengths of 1.347, 1.355, and 1.366 Å for TS 4a, b, and c, respectively. R1 and R2 H-abstractions result in a more stabilized bond length than the reactant bond length of 1.359 Å. The enthalpies of reaction are energetically comparable, suggesting that R-C and γ-C abstraction are thermodynamically competitive. Since the R2 and R3 sites contain the same R group, the enthalpies of reaction for these γ-C H-abstractions are the same. R1 H-abstraction produces an enthalpy of reaction that is only 0.3 kcal mol-1 higher. Examining the product geometries, R1 H-abstraction results in the shortest N-C bond length, 1.340 Å. R2 and R3 H-abstractions have longer N-C bond lengths of

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Figure 5. Reactant, prereactive complex, transition state, and product geometries for N,N-dimethylformamide. Parameters are optimized at the MP2/6-311G(2d,2p) level of theory. Bond lengths are in angstroms and bond angles are in degrees.

1.377 and 1.374 Å, respectively. However, similar to the γ-C H-abstraction for tNMF and cNMF, both R2 and R3 Habstraction in DMF have radical delocalization. This results in reaction enthalpies for R2 and R3 H-abstraction that are as low as R1 H-abstraction. Analysis of DMF shows that R-C and γ-C abstractions are both competitive pathways, yielding similar thermodynamic energies. Kinetically, R-C H-abstraction is more favorable, and despite having the same methyl R group, radical attack prefers attraction at the R2 site over the R3 site.

B. Acetamide Trends. The second group set consists of the acetamides which contain a methyl group at R1. Figure 6 depicts the structures of the parent acetamide (AM) system. Figure 10e shows the energy diagram for acetamide that identifies •OH attack at the R1 site as both the kinetically and thermodynamically favored path, which is similar to the trend observed in FM. The binding energy trends for AM are the same as for FM and tNMF where R3 abstraction is the least favorable, and R2 abstraction can form two H-bonds, making it the most stable.



Figure 6. Reactant, prereactive complex, transition state, and product geometries for acetamide. Parameters are optimized at the MP2/6-311G(2d,2p) level of theory. Bond lengths are in angstroms and bond angles are in degrees.

R1 H-abstraction, the β-C, is the most kinetically favorable with energy differences of 3.4 and 4.2 kcal mol-1 between R2 and R3 H-abstraction, respectively. Again, the trends in kinetic stability correspond to the N-C bond length. R1 H-abstraction results in a shorter bond length than the reactant bond length of 1.371 Å. R2 and R3 H-abstraction have longer and less stable bonds. The kinetic trend confirms the results found in the formamide systems where nitrogen H-abstraction has a relatively high activation energy compared to other abstraction sites. In addition, the acetamide group introduces a new abstraction pathway, β-C H-abstraction, which is more kinetically favorable than nitrogen H-abstraction. The enthalpy of reaction for R1 H-abstraction is -20.1 kcal mol-1, which is 15 kcal mol-1 more stable than the R2 or R3 H-abstraction. The product geometries for R2 and R3 abstraction have N-C bond lengths of 1.412 Å. R1 abstraction is the only

pathway that results in a shorter, more stabilized bond length from the reactant bond length of 1.371 Å. In addition, like γ-C H-abstraction in previous analysis, the radical site in a R1 H-abstraction can resonate between the N-C(O) bonds. The products of R2 and R3 H-abstraction have the same geometry and lack the delocalization of the radical site, resulting in a higher enthalpy of reaction. Thermodynamically, β-C Habstraction is more stable than nitrogen H-abstraction by 15.0 kcal mol-1. 1. trans- and cis-N-Methylacetamide. Analysis of the energy diagrams for cis-N-methylacetamide (cNMA) in Figure 10f and trans-N-methylacetamide (tNMA) in Figure 10g shows addition of a methyl group to the R2 or R3 site leads to a different trend from the formamide set, where the pathway for γ-C Habstraction is now more favorable. Figures 7 and 8 depict the structures of the cNMA and tNMA systems, respectively.

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Figure 7. Reactant, prereactive complex, transition state, and product geometries for cis-N-methylacetamide. Parameters are optimized at the MP2/6-311G(2d,2p) level of theory. Bond lengths are in angstroms and bond angles are in degrees.

Similar to earlier analysis of the trans- and cis-N-methyl systems for the formamide group set, R3 H-abstraction has the highest binding energy in both systems. The lowest binding energy in cNMA is the prereactive complex for R2 H-abstraction due to two H-bonds forming. However, the methyl group at the R2 site in tNMA allows the formation of only one H-bond. As a result, the binding energies for R1 and R2 H-abstraction in tNMA have similar energies. The activation energies in cNMA and tNMA show γ-C H-abstraction is kinetically preferred over nitrogen or β-C H-abstraction. γ-C H-abstraction in cNMA has an activation energy of -3.3 kcal mol-1. This is approximately 3.0 kcal mol-1 lower than R1 or R2 H-abstraction. Between R1 and R2 abstraction, the difference in activation energy is within the root

mean square error of the test cases and therefore, they are considered competitive pathways. The N-C bond lengths in the R1, R2, and R3 TS structures are 1.357, 1.384, and 1.376 Å, respectively. Although R1 H-abstraction has the more stable N-C bond, it has the highest activation energy. This disparity can be attributed to the Hammond postulate, which states that early transitions are more exothermic.45 The distances between the bond broken (i.e., the bond between the carbon and the hydrogen), to the bond forming, (i.e., the bond between the hydrogen and •OH), are 1.220 and 1.240 Å in Ts 6a and 1.167 and 1.396 Å in Ts 6c. Since the hydrogen is closer to the carbon in R3 H-abstraction, it more closely resembles an early transition and, as such, has the lower activation energy. Similarly in tNMA, γ-C H-abstraction results in the lowest activation energy.



Figure 8. Reactant, prereactive complex, transition state, and product geometries for trans-N-methylacetamide. Parameters are optimized at the MP2/6-311G(2d,2p) level of theory. Bond lengths are in angstroms and bond angles are in degrees.

The activation energy for γ-C H-abstraction is 4.1 and 5.2 kcal mol-1 lower than nitrogen and β-C H-abstraction, respectively. The enthalpy of reaction has three distinct energies for the R1, R2, and R3 sites in both cNMA and tNMA. γ-C H-abstraction is thermodynamically the most favorable with an average difference of 5.1 and 12.7 kcal mol-1 between β-C and nitrogen H-abstraction, respectively. Nitrogen H-abstraction has the longest N-C bond length, following the high enthalpy of reaction. The products of β-C and γ-C H-abstractions have

radical delocalization. However, the thermodynamic data and product geometries suggest that there is greater stabilization from resonance in γ-C H-abstraction. The radical methyl-N bond is 1.379 Å, with a difference of 0.068 Å from the reactant’s length for γ-C H-abstraction. In β-C H-abstraction, the radical meth´ from yl-C bond is 1.473 Å, with a difference of only 0.039 Å the reactant’s length. This trend is consistent in tNMA, showing γ-C H-abstraction results in the more stabilized geometry and lower enthalpy of reaction.

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Figure 9. Reactant, prereactive complex, transition state, and product geometries for N,N-dimethylacetamide. Parameters are optimized at the MP2/6-311G(2d,2p) level of theory. Bond lengths are in angstroms and bond angles are in degrees.

Both cNMA and tNMA analyses suggest nitrogen Habstraction lacks resonance stabilization present in both β-C and γ-C H-abstraction and has the highest energies. Despite having the highest binding energy, •OH attacks on the γ-C hydrogen in cNMA and tNMA are kinetically and thermodynamically favored over β-C H-abstraction. 2. N,N-Dimethylacetamide. The N,N-dimethylacetamide (DMA) system further illustrates the competition between γ-C

and R-C abstraction (Figure 10h). Figure 9 depicts the structures of the DMA system. The prereactive complex trends for DMA are the same as those seen in cNMF, DMF, and tNMA. R3 abstraction has the highest binding energy, while R1 and R2 abstraction have lower binding energies and are cocompetitive with each other. The kinetics of the DMA system differs from that of the other systems. In the transition state, R1 abstraction, which tends to


Figure 10. Potential energy surface for reaction with model amides at 0 K (kcal mol-1).


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TABLE 3: Relative Energetic for Hydrogen Abstraction by the OH Radical for Model Amides at 0 K (kcal mol-1)a R1 system formamide

trans-N-methylformamide

cis-N-methylformamide

N,N-dimethylformamide

acetamide

cis-N-methylacetamide

trans-N-methylacetamide

N,N-dimethylacetamide

a

R2

R3

method

precmpx

Eact

∆Hrxn

precmpx

Eact

∆Hrxn

precmpx

Eact

∆Hrxn

UMP2/6-31G(d) UMP2/6-311G(2d,2p) CCSD(T)/6-311G(2d,2p) CCSD(T)/6-311++G(2df,2p) G2 CBS-Q UMP2/6-31G(d) UMP2/6-311G(2d,2p) CCSD(T)/6-311G(2d,2p) CCSD(T)/6-311++G(2df,2p) G2 CBS-Q UMP2/6-31G(d) UMP2/6-311G(2d,2p) CCSD(T)/6-311G(2d,2p) CCSD(T)/6-311++G(2df,2p) G2 CBS-Q UMP2/6-31G(d) UMP2/6-311G(2d,2p) CCSD(T)/6-311G(2d,2p) CCSD(T)/6-311++G(2df,2p) G2 CBS-Q UMP2/6-31G(d) UMP2/6-311G(2d,2p) CCSD(T)/6-311G(2d,2p) CCSD(T)/6-311++G(2df,2p) G2 CBS-Q UMP2/6-31G(d) UMP2/6-311G(2d,2p) CCSD(T)/6-311G(2d,2p) CCSD(T)/6-311++G(2df,2p) G2 CBS-Q UMP2/6-31G(d) UMP2/6-311G(2d,2p) CCSD(T)/6-311G(2d,2p) CCSD(T)/6-311++G(2df,2p) G2 CBS-Q UMP2/6-31G(d) UMP2/6-311G(2d,2p) CCSD(T)/6-311G(2d,2p) CCSD(T)/6-311++G(2df,2p) G2 CBS-Q

–5.4 –7.3 –7.0 –5.2 –7.2 –5.3 –5.7 –7.7 –7.5 –5.7 –7.8 –6.0 –5.7 –7.6 –7.4 –5.6 –7.9 –5.9 –6.0 –8.0 –7.7 –6.0 –8.2 –6.2 –6.6 –8.4 –8.1 –6.3 –8.3 –6.4 –6.8 –8.7 –8.5 –6.5 –8.8 –6.9 –6.7 –8.5 –8.3 –6.4 –8.8 –6.8 –6.8 –8.8 –8.6 –6.7 –9.0 –7.1

7.8 2.5 –1.6 –1.3 –4.2 –4.4 7.3 1.8 –2.4 –1.9 –4.8 –5.1 7.3 2.1 –2.2 –1.9 –4.8 –5.2 7.1 1.6 –2.6 –2.3 –5.0 –5.5 9.9 4.0 3.0 2.8 0.6 0.1 9.5 3.4 2.5 2.3 0.3 –0.4 9.6 3.8 2.7 2.6 0.0 –0.5 9.0 3.1 2.1 1.8 –0.5 –1.1

–18.5 –26.7 –22.6 –23.7 –24.8 –25.0 –18.4 –26.4 –22.4 –23.7 –25.1 –25.2 –17.9 –25.8 –21.8 –23.2 –24.6 –24.6 –17.8 –25.5 –21.6 –23.1 –24.6 –24.7 –10.7 –18.3 –15.6 –18.5 –19.9 –20.1 –10.4 –18.2 –15.7 –18.4 –20.1 –20.5 –11.2 –18.8 –15.9 –18.6 –20.0 –20.3 –10.3 –18.0 –15.4 –18.1 –19.5 –20.0

–4.8 –6.5 –6.4 –5.3 –7.3 –5.5 –8.4 –10.0 –9.9 –7.3 –9.4 –7.4 5.8 –7.7 –7.6 –5.5 –7.8 –5.8 –5.9 –7.8 –7.7 –5.8 –8.1 –6.1 –8.5 –10.2 –9.9 –7.6 –9.6 –7.5 –8.9 –10.6 –10.4 –7.9 –10.0 –8.1 –6.2 –8.0 –7.9 –6.0 –8.3 –6.4 –6.1 –8.0 –7.9 –6.2 –8.5 –6.6

14.1 9.0 6.4 7.7 5.0 5.0 10.8 5.9 3.1 4.0 1.3 1.0 7.2 1.6 –0.3 –0.7 –3.2 –3.7 7.5 1.8 –0.2 –0.8 –3.4 –4.0 12.6 7.7 5.2 6.3 3.7 3.5 9.3 4.4 1.6 2.5 –0.1 –0.7 6.5 0.9 –1.0 –1.6 –4.1 –4.6 6.2 0.7 –0.7 –2.1 –4.8 –5.5

3.4 –2.6 –2.8 –3.0 –4.2 –3.6 6.9 –7.5 –8.2 –9.3 –9.0 –10.4 –15.2 –24.2 –21.0 –23.7 –24.3 –24.5 –14.9 –24.5 –21.1 –24.0 –24.6 –25.0 1.1 –4.4 –4.8 –5.4 –6.7 –5.1 6.6 –10.9 –11.7 –12.8 –14.5 –14.0 –15.6 –24.4 –21.1 –23.8 –24.3 –24.6 –15.2 –24.1 –22.8 –24.0 –26.3 –25.2

–1.5 –3.8 –3.8 –2.4 –4.2 –2.5 –2.3 –3.3 –3.2 –1.5 –3.4 –1.0 –1.8 –3.9 –3.8 –2.5 –4.9 –2.9 –2.3 –4.1 –3.5 –1.7 –4.1 –1.7 –1.6 –4.0 –3.9 –2.3 –4.5 –2.5 0.6 –4.9 –4.0 –2.1 –4.6 –2.4 –1.9 –4.0 –4.0 –2.4 –4.8 –2.9 –3.5 –4.9 –4.7 –2.5 –5.1 –3.0

17.3 10.1 8.5 9.9 5.1 5.5 9.6 3.9 1.8 0.9 –1.3 –2.2 13.0 5.9 4.5 5.2 2.1 1.8 9.0 3.1 0.5 0.5 –2.1 –2.9 14.2 7.2 5.6 6.9 3.5 4.3 8.0 2.2 –0.5 0.0 –2.5 –3.3 10.5 3.4 2.1 3.0 0.9 0.6 8.4 2.2 –0.7 –0.5 –3.1 –4.0

3.5 –2.6 –2.8 –2.6 –4.2 –3.6 –15.8 –24.4 –21.2 –23.6 –24.6 –24.8 –1.0 –6.1 –7.0 –8.0 –9.7 –8.9 –15.3 –24.0 –21.3 –23.8 –24.6 –25.0 1.1 –4.4 –4.8 –5.4 –6.7 –5.1 –16.8 –25.7 –22.5 –25.0 –25.8 –26.3 –2.8 –8.4 –9.2 –10.4 –12.2 –11.5 –16.7 –25.8 –22.8 –25.7 –26.3 –26.9

Includes UMP2(full)/6-311G(2d,2p) zero-point corrections (excludes G2 and CBS-Q).

be lowest in energy, has the highest activation energy. R2 abstraction is now the most kinetically favorable, with an activation energy that is 1.5 and 4.4 kcal mol-1 lower than R3 and R1 H-abstraction, respectively. The trends in bond lengths of DMA have the same disparity mentioned in tNMA and cNMA. However, application of the Hammond postulate along with hydrogen bonding resolves the disparity and predicts that R2 H-abstraction will be the most stable, following activation energy trends. Although the TS 8a has a strong H-bond, it is an intermediate transition state, making it less stable than the TS 8b and 8c which are earlier transition states. Between R2 and R3 H-abstraction, however, the •OH has a slight preference for attack at the R2 site as a result of stabilization by hydrogen bonding. The thermodynamic trends also differ from most of the trends seen in the previous formamide analysis. R3 abstraction is the most stable at -26.9 kcal mol-1 which is 1.7 and 6.9 kcal mol-1 lower in energy than R2 and R1 abstraction, respectively. Despite having the same methyl R group, abstractions off the R2 and R3 site do not have the same product geometries. This could be

due to reduction in steric interaction with the R1 methyl group when an R3 methyl hydrogen is removed. The •OH prefers attack on the γ-C, both kinetically and thermodynamically, rather than the β-C. However, analysis of DMA, also, shows H-abstraction off the R2 site is slightly more kinetically favored while the R3 site is slightly more thermodynamically favored when R2 and R3 have the same methyl R group and the R1 site is methylated. C. Group Trends among the Amides. 1. Formamide Group. The formamide group data set shows R-C H-abstraction is kinetically and thermodynamically favored over nitrogen H-abstraction. In FM, the difference in activation energy is approximately 10.0 kcal mol-1. For tNMF and cNMF, R-C H-abstractions have lower activation energy than nitrogen H-abstraction by 6.1 and 7.0 kcal mol-1, respectively. There is an immense difference in reaction enthalpy between the two abstraction sites as well. R-C H-abstraction is lower in energy than nitrogen H-abstraction by 31.5, 14.8, and 15.7 kcal mol-1 for FM, tNMF, and cNMF, respectively. However, an interesting change occurs when a methyl group is present in the system.

Reaction of OH Radicals with Peptide Systems Comparison of FM with tNMF and cNMF shows a clear change in the activation energy and enthalpy of reaction for nitrogen H-abstraction. In FM, the activation energies for the two sites of nitrogen H-abstraction are 5.5 and 5.0 kcal mol-1. With an additional methyl group on the nitrogen, the activation energy decreases by approximately 4.0 kcal mol-1. The enthalpy of reaction between FM and the trans and cis NMF for nitrogen H-abstraction also decreases by a significant amount, 6.8 and 5.3 kcal mol-1, respectively. The large decrease in the nitrogen H-abstraction pathways with an additional methyl group makes it more competitive with R-C H-abstraction. However, for all the formamide systems, R-C H-abstraction continues to be both the kinetically and thermodynamically favored pathway. The energy diagrams for tNMF, cNMF, and DM, shown in Figure 10b-d, demonstrate a close competition between R-C and γ-C H-abstraction. Although the activation energy remains lowest for R-C H-abstraction for these systems, γ-C Habstraction is consistently lower in energy than nitrogen H-abstraction by 5.5 and 3.2 kcal mol-1 in tNMF and cNMF. While there is a significant difference between γ-C and nitrogen H-abstraction, the difference between R-C and γ-C H-abstraction is only 2.9 and 1.5 kcal mol-1 in the same two systems. For DMF, the difference in activation energy between R-C and γ-C H-abstractions, at either the R2 or R3 sites, is approximately 2.0 kcal mol-1. In all formamide systems, the differences in enthalpies of reaction for R-C and γ-C H-abstractions are within the root mean square error, making them cocompetitive pathways. For example, in cNMF, the difference in enthalpies of reaction for R-C and γ-C H-abstraction is only 0.1 kcal mol-1. 2. Acetamide Group. In the acetamide group set, β-C H-abstraction occurs on a methyl group bounded to the carbonyl group and R-C H-abstraction is not possible. β-C H-abstraction has a lower activation energy and enthalpy of reaction than nitrogen H-abstraction in the first three acetamide systems. As seen in the formamide group set, the nitrogen H-abstraction pathway demonstrates increased stabilization both kinetically and thermodynamically with an additional methyl group at the R2 or R3 site. The activation energy of nitrogen H-abstraction in cNMA and tNMA is approximately 4.0 kcal mol-1 lower than that in AM, the same decrease seen in the formamide group data. The same trend appears in AM where the enthalpy of reaction for nitrogen H-abstraction decreases by 9.0 and 6.4 kcal mol-1 from AM to cNMA and tNMA, respectively. When the energy diagrams of cNMA, tNMA, and DMA are compared, the energies show that γ-C H-abstraction becomes the preferential pathway for radical attack over β-C Habstraction. The activation energies for γ-C H-abstraction are 2.6 and 4.1 kcal mol-1 lower than β-C H-abstraction in cNMA and tNMA. For DMA, this trend is also observed; R3 and R2 γ-C H-abstraction is 2.9 and 4.4 kcal mol-1 lower, respectively, than β-C H-abstraction. The enthalpies of reaction follow the same trend as well. In cNMA and tNMA, γ-C H-abstraction is lower by 5.8 and 4.3 kcal mol-1, respectively. In DMA, the difference is 6.9 and 5.2 kcal mol-1. As a result, it can be concluded that γ-C H-abstraction will be more favorable, both kinetically and thermodynamically, than either β-C or nitrogen H-abstraction. 3. OWerall Trends. Overall trends between the eight amides show nitrogen H-abstraction is not preferred for either the R2 or R3 sites. This pathway has the highest enthalpies of reaction in all six cases where it is available, and it has the highest activation energy in five out of the six cases with this structure arrangement. The anomaly was in cNMA where β-C Habstraction was higher in energy by -0.3 kcal mol-1; this

J. Phys. Chem. A, Vol. 114, No. 16, 2010 5355 difference is within the 1.1 kcal mol-1 error determined in the calibration test. Therefore, the nitrogen H-abstraction pathway remains the least preferred overall. As mentioned in earlier analysis, methylation at R2 and R3 noticeably lowers the activation energy and enthalpy of reaction for nitrogen H-abstraction. However, comparison between the data sets of the parent systems, FM and AM, shows that methylation of the carbonyl group, also, stabilizes nitrogen H-abstraction significantly, while γ-C H-abstraction does not change significantly between the two group systems. For example, the activation energy for nitrogen H-abstraction from FM to AM decreases on average by 1.3 kcal mol-1. In all cases where nitrogen H-abstraction is possible, the acetamide group set provides the lowest energies. Therefore, a methyl group on the carbonyl group causes nitrogen H-abstraction to be more competitive with the other pathways as well as methylation at the R2 or R3 site. Another trend seen between the formamide and acetamide group sets is H-abstraction off the R3 site consistently has the highest prereactive complex energy due to poor hydrogen bonding. R1 and R2 abstraction have lower binding energies than R3 abstraction. When the R2 group is a hydrogen, the binding energy for R2 abstraction is lowest. This is seen in parts a, b, e, and f of Figure 10 for FM, tNMF, AM, and cNMA. When the R2 group is a methyl, the prereactive complexes for R1 and R2 abstraction are similar. The presence of the methyl group at the R2 site prevents formation of a secondary H-bond that occurs in the previously mentioned groups. This is seen in parts c, d, g, and h of Figure 10 for cNMF, DMF, tNMA, and DMA. The binding energy of the prereactive complex contributes to the favorability or unfavorability of the abstraction pathway, but in the amide systems there is no one site which has the lowest binding energy within root mean square deviation of the test cases to determine absolute preference. Comparison of the formamides and acetamides also shows the differences between R-C and β-C H-abstraction. R-C H-abstraction has a lower activation energy and enthalpy of formation than β-C H-abstraction as seen in comparison of parts a and e of Figure 10. There is a 4.6 kcal mol-1 difference in transition state energies and a 4.9 kcal mol-1 difference in products stabilities between formamide and acetamide Habstraction at the R1 site. This difference is consistent between the other systems as well. IV. Discussion There have been many studies that have looked into •OH attack on proteins, particularly amino acids.10,18-21 Some studies appear to conflict with parts of the findings from this study. Stefanic et al. determined with empiricial evidence, through pulse radiolysis on glycine and a glycine anion, that the nitrogen site is the preferred site of •OH attack.19 Liesmann et al., confirms the same preference with experimental and theoretical H-abstraction on L-alanine and L-alanine ethyl ester.21 Their results disagree with the findings from this study which states that the N-site is the least preferential in •OH attack compared to R-C, β-C, or γ-C abstraction. The key difference that accounts for the contradiction is that this study models a peptide bond, where two amino acids are joined, while the other studies investigated isolated amino acids. Another key difference to note is the R-hydrogen in the amide systems is not present in an amino acid and the R-C in proteins and amino acids is equivalent to the β-C and γ-C in the amide models. The findings for these two abstractions are consistent with earlier studies which concluded that the most common

5356


pathway for •OH attack was H-abstraction off the R-C for an amino acid.19-22 The R-C is consistently a competitive site of • OH attack. From the model amide systems, γ-C H-abstraction is a competitive pathway for •OH attack with β-C H-abstraction slightly less favorable, but more favorable than nitrogen H-abstraction. Since the amino acid R-C is equivalent to the amide model γ-C and β-C, the findings are consistent. The trends seen in this study have major implications in the study of large polypeptide chains and small proteins. When these results are applied to a polypeptide chain, the computational study of protein folding, specifically an ab initio approach, is less time intensive and costly. Rather than having to study an entire polypeptide chain, the trends can be used to identify and locate specific sites within the chain where radical attack is likely to occur. With the area of study narrowed to a small section, this drastically reduces the computational power needed when looking at protein folding and misfolding that occur as a result of oxidative stress. Molecular dynamics (MD) simulations have already shown a promising way to explore the protein folding problem.50-52 However, MD simulations uses parametrizations which decrease the reliability and accuracy of the calculations. Ab initio is based on first principles and therefore provides a more accurate model. Reducing the protein to a smaller model which displays the preferential site of H-abstraction by the •OH offers an alternative and promising way to predict the location of radical damage and possible misfolding for small proteins using ab initio methods, which is a more accurate calculation of radical energy.50 This study serves as a strong foundational analysis for continued research in this area. Further ab initio investigation will use a larger model system, one with more of a resemblance to the protein backbone. The larger model system will be in better agreement with previous studies’ systems which may provide further insight into the disparity mentioned above between this study and previous results. The amide systems used in this study only observes the peptide bond in a protein backbone. The next step will be to model a derivative of the amino acid glycine which contains two peptide bonds and serves as a better model of a polypeptide chain environment. V. Conclusion The findings from this study show that preferential •OH attack will occur at the R-C site for formamide. The addition of a methyl group on the nitrogen results in an alternative, competitive site for •OH attack, the γ-C site; which is competitive with R-C H-abstraction in the latter three formamide systems. In the acetamide systems, γ-C H-abstraction is both kinetically and thermodynamically favored over β-C abstraction. Additional analysis shows an interesting phenomenon occurring in the nitrogen H-abstraction pathway for all systems where it occurs. Methylation of any of the three R site results in a significant stabilization of both the transition states and the products during H-abstraction. Nitrogen H-abstraction is consistently disfavored for all systems with the highest activation energies and enthalpies of reaction. Methylation, however, makes it more competitive with the other pathways. Acknowledgment. We would like to thank the Rosen Center for Advanced Computing at Purdue University for computational resources to complete this work. Supporting Information Available: Tables of coordinates for the compounds discussed. This material is available free of charge via the Internet at http://pubs.acs.org.

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JP100375C

Primary Steps in the Reaction of OH Radicals with Peptide Systems

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