Chain-Breaking Activity of Carotenes in Lipid Peroxidation: A

J. Phys. Chem. B 2009, 113, 15699–15708

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Chain-Breaking Activity of Carotenes in Lipid Peroxidation: A Theoretical Study Jian-Jhih Guo, Hong-Yi Hsieh, and Ching-Han Hu* Department of Chemistry, National Changhua UniVersity of Education, Changhua 50058, Taiwan ReceiVed: August 13, 2009; ReVised Manuscript ReceiVed: October 6, 2009

Chain-breaking reactions against lipid peroxidation performed by carotenes, including β-carotene (β-CAR) and lycopene (LYC), have been studied using density functional theory. We chose linoleic acid (LAH) as the lipid model and examined two mechanisms: hydrogen abstraction and addition. Our computed reaction diagrams reveal that the addition mechanism is able to offer a larger extent of chain-breaking protection than hydrogen abstraction. In the case of hydrogen abstraction, the resulting carotene radical CAR(-H)• has a smaller O2 affinity than the linoleic acid radical (LA•). Formation of the addition adduct radical ROO-CAR• is energetically favorable, and it has an even smaller tendency to react with O2 than CAR(-H)•. Comparatively, ROO-βCAR• is less likely to react with O2 than ROO-LYC•. Both the hydrogen abstraction and addition radicals (CAR(-H)• and ROO-CAR•) react readily with a second ROO• radical via either hydrogen abstraction or addition. 1. Introduction Lipid peroxidation is a free radical chain reaction involving several stages. The first stage is the initiation (eq 1), in which an initiator generates the radical species through hydrogen abstraction. In the presence of oxygen, a peroxyl radical is generated (eq 2), followed by hydrogen abstraction from a nearby lipid (eq 3). With constant supply of O2, this propagation step oxidizes the lipid unless this chain reaction is terminated. For example, the formation of tetraoxide by combination of two peroxyl radicals is given in eq 4. •

cigarette smoke oxidants it does not amplify lipid oxidations in biologically relevant liposome membranes.17 Despite the aforementioned concerns of individual supplementary carotenoids, consumption of fruits and vegetables rich in carotenoids is still recommended.14,18 Several mechanisms for the antioxidative function of carotenoid have been suggested. These mechanisms include charge transfer, hydrogen transfer, and addition (adduct formation)1,19

Electron transfer: CAR + ROO• f CAR•+ + ROO-

(5)

(1)

Initiation: RH f R

Hydrogen abstraction: CAR + ROO• f CAR(-H)• + ROOH •

•

Propagation: R + O2 f R-OO

(2)

ROO• + RH f ROOH + R•

(3)

Termination: ROO• + ROO• f ROO-OOR

(4)

Chain-breaking antioxidants react with the peroxyl radical, thus terminating the propagation of peroxidation. Carotenoid is a type of compound that is considered to exhibit chain-breaking activity.1-12 The protective functions of carotenoids have been attributed to their polyene frameworks. Among the carotenoids, β-carotene (β-CAR) has attracted the highest attention as a chain-breaking antioxidant.11,12 β-CAR was shown to be an efficient scavenger of peroxyl radicals at low oxygen pressure; however, it becomes pro-oxidative at high oxygen pressure.1,13 There have been observations of adverse effects of supplemental β-CAR in the case of smokers and workers exposed to asbestos.14,15 On the basis of experiments on liposome and microsome membranes, Liebler et al. reported that β-CAR as an antioxidant is ineffective in preventing lipid peroxidation.16 Nevertheless, a later experiment of Liebler et al. revealed that although β-CAR undergoes auto-oxidation in the presence of * Corresponding author. E-mail: [email protected].

(6)

Addition: CAR + ROO• f ROO-CAR•

(7)

Another noteworthy mechanism proposed for the carotenoid function involves the formation of carotenoid aggregates.19,20 Aggregate formation is suggested to explain the fact that CAR loses its antioxidative activity or that it becomes pro-oxidative at high concentrations. Computational studies concerning the antiradical properties of carotenoids have been performed.21-24 The capacity of a carotenoid species was evaluated in terms of their electron donating power21-23 and by their C-H bond dissociation energies (BDEs).24 While these studies have provided valuable information for the properties of carotenoids, we wish to investigate the mechanism involved in the chain-breaking functions exhibited by carotenoids. Product analysis of radical-carotenoid reactions reveals that the reaction mechanism depends on the nature of the radical species and the environment where the reactions occur. For carbon-centered peroxyl radicals, the electron transfer mechanism is proposed to be the dominant pathway involved in the reactions of peroxyl radicals with carotenoids in aqueous micelles.25 In contrast, within nonpolar environments, electron transfer is less favored.19,26

10.1021/jp907822h CCC: $40.75  2009 American Chemical Society Published on Web 11/03/2009

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SCHEME 1: Structure and Numbering Schemes for Carotenes Investigated in This Study: β-Carotene (β-CAR) and Lycopene (LYC)

Hydrogen transfer has been proposed to be involved in the reaction between a peroxyl radical and carotenoid.27-31 However, adduct formation (addition) has been suggested to be the more likely mechanism than hydrogen abstraction in nonpolar environments.1,26,32 On the basis of experiments performed in nonpolar solvents, Burton and Ingold suggested that β-CAR scavenges the peroxyl radical via formation of addition adduct ROO-β-CAR• (eq 7).1 The proposed mechanism was offered to explain the observation that at high oxygen pressure β-CAR becomes pro-oxidative. In this proposal, an auto-oxidative process is involved in the presence of oxygen, resulting in hydrogen abstraction

ROO-CAR• + O2 f ROO-CAR-OO•

(8)

ROO-CAR-OO• + RH f ROO-CAR-OOH + R• (9) The affinity to oxygen for many of the adduct radicals has been measured in different environments.26,33 El-Agamey and McGarvey have given evidence for a lack of reactivity of carotenoid addition radicals toward oxygen, based on laser flash photolysis study for the reactions of acylperoxyl radical-carotenoid adducts in polar and nonpolar solvents.26 In nonpolar solvents, the addition radical was observed to decay according to firstorder kinetics, indicating that the radical does not react with O2.26 The authors later discovered reversible addition of the ROO-CAR• radical with O2, when a carotenoid compound of shorter π-conjugation chain is used.33 It was suggested that the radical addition product (of eq 8) would react with another peroxyl radical to form ROO-CAR-OOR (eq 10)19,34 or ROOH + ROOCAR(-H) (eq 11).

ROO-CAR• + ROO• f ROO-CAR-OOR

(10)

ROO-CAR• + ROO• f ROOH + ROO-CAR(-H) (11) Hydrogen abstractions from the lipid by ROO-CAR-OO• or CAR(-H)-OO• are exothermic, thus a chain-breaking antioxidant is expected to have low O2 affinity. We aim to investigate the reactions between peroxyl radicals and carotenoids in the presence of lipid and O2. We included two important C40H56 carotenes (carotenoids consisting of only C and H atoms): β-carotene and lycopene (β-CAR and LYC, Scheme 1). β-CAR and LYC are the most abundant carotenoids

in human plasma. Assuming that these compounds would function in the less polar region of biological systems where the electron transfer mechanism is less important, we focus on two of the aforementioned mechanisms, i.e., hydrogen abstraction and addition. 2. Computational Approach Density functional theory (DFT) was applied in this study. The C-H BDEs (enthalpies at 298 K) of CH3-H and CH2dCHCH2-H, OO-H BDEs of CH3OO-H and CH2d CHCH2OO-H, O2 affinities of CH3• and CH2dCHCH2•, and enthalpy for the addition of CH3OO• to CH2dCH-CHdCH2 were examined with various functionals and were compared with those obtained from high-level ab initio theory. The tested functionals include pure, generalized gradient approximation (GGA) methods including BLYP,35,36 BPW91,35,37 and PBEPBE.38 We have also ascertained the accuracy of hybrid GGAs (B3LYP36,39 and MPW1PW91),37,40 the kinetic energy density dependent meta-GGA functionals (BB95,41 MPW1K,40,42 MPW1B95,40,41 and MPWB1K),43 and the second-order perturbation energy-dependent double-hybrid GGA functionals (B2PLYP, MPW2-PLYP).44,45 Three basis sets, including 6-31G(d), 6-31++G(d,p), and 6-311++G(3df,3pd) were included in the DFT computations. Geometry optimizations were performed with the 6-31G(d) basis set, and the harmonic vibrational frequencies were used to obtain the thermal corrections to enthalpies at 298 K. In the case of radical species, both unrestricted and restricted open-shell (RO) schemes were applied. A computational strategy resulted from the systematic comparisons of DFT with the high-level ab initio G346 method. The computational strategy will be used in the reactions of β-CAR and LYC with peroxyl radicals. For the lipid model, SCHEME 2: Structure and Numbering Scheme for Linoleic Acid (LAH)

Chain Breaking of Carotenes in Lipid Peroxidation

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Figure 1. Average absolute deviations (AADs) for the prediction of seven tested reactions using various DFT methods. Reaction enthalpies obtained with G3 theory were used as the reference. In (a) and (b), geometries were obtained with B3LYP/6-31G(d), while in (c) and (d), the geometries were, respectively, optimized with individual functionals. Basis sets are: A, 6-31G(d); B, 6-31++G(d,p); and C, 6-311++G(3df,3pd). See Supporting Information for detailed data.

we used linoleic acid (LAH, Scheme 2). The computations were performed using the Gaussian03 series of programs.47 3. Results and Discussion Test Computations of Various DFT Methods. DFT has been recognized to underestimate the BDEs of C-H and OO-H bonds.48,49 To better define our methodology, we performed computations on model systems using DFT. The energetics obtained using various DFT functionals were compared with those obtained using the G3 level of theory. The average absolute deviations (AADs) of DFT predictions using three basis sets are illustrated in Figure 1. The performance of DFT computed at the B3LYP/6-31G(d) optimized geometries is shown in Figures 1a and 1b, while those computed using optimized geometries from individual functionals are shown in Figures 1c and 1d. As shown in the figures, the use of B3LYP/

6-31G(d) optimized geometries has a minimum influence on the accuracy of energetics. Generally, the AADs are reduced when larger basis sets are applied. The improvement due to basis set quality is more significant with the restricted open-shell (RO) scheme. The MPWB1K functional with 6-31++G(d,p) has the smallest AAD among all tested methods. The accuracy of MPWB1K is higher than its RO counterpart, i.e., ROMPWB1K. This observation is inconsistent with the recent study of Radom et al., in which the restricted version was demonstrated to provide more promising results for the C-H BDEs.50 The better performance of MPWB1K is attributed to its improved description for the BDEs of CH3-OO•, CH2dCHCH2-OO•, and CH3OOCH2CHdCHCH2• (see Supporting Information). The double-hybrid functionals (B2-PLYP and MPW2-PLYP) developed by Grimme et al. have been demonstrated to be

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TABLE 1: C-H BDEs, Enthalpy of O2 Addition to Carbon Centered Radicals, and OO-H BDEs of β-CAR, LYC, and LAHc

and LYC have been located; however, they are slightly higher in energy than their C1 counterparts (see Supporting Information). The carotene radicals β-CAR(-H)• and LYC(-H)• are also possible targets of hydrogen abstraction from a nearby radical. The weakest C-H bond occurs on C3 (refer to Scheme 1). The second C-H BDEs of β-CAR and LYC [forming β-CAR(-H2) and LYC(-H2)] are significantly lower than the first C-H BDEs. In the case of β-CAR(-H)•, C-H BDE is 61.6 kcal/ mol, and in the case of LYC(-H)•, C-H BDE is remarkably lowered to 52.5 kcal/mol. The extraordinarily low C-H BDE of LYC(-H)• is attributed to its extended π-delocalization with the hydrogen abstraction at C3. There are 13 conjugated double bonds in LYC(-H2), while in β-CAR(-H2) there are 12 conjugated double bonds. The results suggest that when O2 concentration is very low LYC is more capable of quenching radicals via two consecutive hydrogen donations than β-CAR. The first step for the propagation of a lipid peroxidation involves the addition of the initiated radical toward oxygen (eq 2). Therefore, the reactivity of the peroxyl-carotene radical toward O2 has been a subject of interest.26 O2 affinity for the methyl radical is the largest (32.3 kcal/mol), and the affinities of the model allyl and biallyl radicals are reduced to 18.0 and 10.6 kcal/mol, respectively. The O2 affinity of LA• is similar (12.2 kcal/mol) to that of the model biallyl radical and is larger than those of carotenes. In contrast, the lactone species demonstrate a much smaller tendency toward O2 addition. This feature has been referred to as the antioxidative capacity of lactones.51 O2 affinities of β-CAR(-H)• and LYC(-H)• (3.5 and 3.1 kcal/mol, respectively) are even smaller than those of lactones. The low affinity of extended π-systems toward O2 has been demonstrated by the theoretical study of Porter et al.48 The computed OO-H BDE ranges from 79.2 to 82.5 kcal/ mol (Table 1), a much smaller window compared with C-H BDE and O2 affinity. Furthermore, for LAH and carotene systems, OO-H BDEs are more than 10 kcal/mol higher than C-H BDEs. Therefore, the peroxyl radical is more likely to undergo hydrogen abstraction from the surroundings than carbon-centered radicals. In the case of LAH (Scheme 2), the lowest-energy path of peroxidation occurs through C-H dissociation on C11, followed by O2 addition and hydrogen abstraction of the peroxyl radical on C13. Our observation is in agreement with the conclusion of the theoretical study of Eriksson et al., in which nona-3,6diene was used as the model for LAH.52 Adduct Formation (Addition). In contrast to hydrogen abstraction (eq 6), the peroxyl radical can add to carotene and form the addition adduct ROO-CAR• (eq 7). According to Burton and Ingold, it is the ROO-CAR• radical that exhibits the chain-breaking function.1 The radical could add to the π-conjugated systems of carotenes in several possible ways. As shown in Table 2, the addition of peroxyl radical CH3OO• to C5 on β-CAR and LYC is the most exothermic. Enthalpies for

a C-H BDE on C4′ is 61.6 (62.4) kcal/mol. b C-H BDE on C4′ is 61.5 (60.9) kcal/mol. c Only species involved in the minimum energy path are presented. Refer to Supporting Information for a complete survey. Data computed using MPWB1K/6-31++G(d,p)// B3LYP/6-31G(d) are in parentheses.

capable of offering accurate predictions for the thermodynamics of molecules.44,45 However, in our test cases, these functionals are not more accurate than GGA or meta-GGA methods. Similar to previous theoretical study,48 our data show that DFT underestimates the BDEs of C-H and OO-H. For example, MPWB1K/6-31++G(d,p) underestimates BDEs of CH2dCHCH2-H and CH2dCHCH2OO-H from G3 theory by 2.5 and 5.9 kcal/mol, respectively (see Supporting Information). Despite the deviation of BDE predictions, the computed reaction energies are expected to be more accurate owing to cancellation of error in these systems.49 Considering the computational cost and efficiency, we used B3LYP/6-31G(d) in all computations. For important species, MPWB1K/6-31++G(d,p) single-point calculations were followed. Unless otherwise stated, we use MPWB1K/6-31++G(d,p) energies in the following discussions. C-H BDE, O2 Affinity of Radicals, and Hydrogen Abstraction of Peroxyl Radicals. The predicted C-H BDEs of carotenes and the LAOO-CAR• adducts are summarized in Table 1. For comparison, we computed C-H BDEs of methane, model alkenes, and lactones. Lactones have been recognized as an efficient type of chain-breaking reagent.51 The C-H BDEs of β-CAR and LYC (the lowest being at C4) are noticeably smaller than those of alkenes and lactones. In addition, C-H BDEs of β-CAR and LYC are slightly smaller than those of LAH. Thus, both carotenes are able to donate a hydrogen atom to LA•. The minima of β-CAR and LYC are of C1 (∼C2) symmetry. Symmetric geometries of Ci symmetry for β-CAR

TABLE 2: Enthalpies of Adduct Formation for the Addition of ROO• to Various Locations of β-CAR and LYCa C5

C6

C7

R ) CH3 R ) LA

-20.4 (-23.5) -18.9 (-22.0)

4.1

-17.3

R ) CH3 R ) LA

-22.5 (-25.8) -18.1 (-21.3)

6.5

-14.5

a

C8

C9

C10

C11

ROO• + β-CAR f ROO-β-CAR• -2.7 -12.5 -5.2 -11.0 ROO• + LYC f ROO-LYC• -1.2 -13.2 -5.2

Data computed using MPWB1K/6-31++G(d,p)//B3LYP/6-31G(d) are in parentheses.

-12.0

C12

C13

C14

C15

-8.1

-10.0

-9.2

-9.5

-7.3

-11.6

-10.2

-11.0



Figure 2. Structures and selected bond distances (in angstroms) of (a) CH3OO-β-CAR• and CH3OO-β-CAR-OO• (5-5′ and 5-6) and (b) CH3OO-LYC• and CH3OO-LYC-OO• (5-5′ and 5-6). The numbers represent locations of CH3OO addition and O2 addition, respectively.

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TABLE 3: C-H BDEs of (C5) ROO-β-CAR• and ROO-LYC• at Various Locationsb

C19

C20

C20′

C19′

C18′

R ) CH3 R ) LA R ) CH3 R ) LA

C4′

ROO-β-CAR f ROO-β-CAR(-H) + H 94.5a 97.2a 71.5 69.9 65.2 60.3 (61.3) 61.3 (62.1) •

•

•

ROO-LYC• f ROO-LYC(-H)• + H• 97.1a 99.4a 74.6 74.1 68.2

61.7 (61.2) 61.2 (60.9)

a Methylene cyclopropane structure resulted. b Data computed using MPWB1K/6-31++G(d,p)//B3LYP/6-31G(d) are in parentheses.

the addition of CH3OO• on β-CAR and LYC are -23.5 and -25.8 kcal/mol, respectively. We have located the minimum of LAOO-β-CAR• and LAOO-LYC• on the C5 position. Enthalpies for the addition of LAOO• to β-CAR and LYC on C5 are -22.0 and -21.3 kcal/mol, respectively. Comparatively, LAOO• forms weaker bonds with carotenes than CH3OO•. Similarly, in forming ROO-CAR-OOR, LAOO• forms weaker bonds with ROOCAR• than CH3OO• (vide infra). The structures of CH3OO-βCAR• and CH3OO-LYC• (CH3OO on C5) and CH3OO-βCAR-OO• and CH3OO-LYC-OO• (O2 on C6 and C5′) are illustrated in Figure 2. For the (C5) CH3OO-CAR• radicals, allyl C-H dissociations at relevant locations have been investigated, and the results are summarized in Table 3. The smallest C-H BDE occurs on C4′. The C-H BDEs on C18′, C19′, and C20′ are several kilocalories per mole higher in energy, which can be attributed to the lack

of π-delocalization in their resulting compounds. The BDEs for C19 and C20 are extremely large, and their geometry optimizations result in methylene cyclopropane species. This C-H BDEs of CH3OO-β-CAR• and CH3OO-LYC• at C4′ (61.3 and 61.2 kcal/mol, respectively) are similar to that of β-CAR(-H)• and are significantly smaller than those of LAH, β-CAR, and LYC. The computed data are in concert with the proposal of Burton and Ingold that the peroxyl radical-carotene adduct, being a good hydrogen donor, may be the chain-breaking species.1 C-H BDEs of the LAOO-CAR• adducts are close to those of CH3OO-CAR•. Enthalpies of O2 addition (eq 8) and corresponding OO-H BDEs of CH3OO-CAR• are summarized in Table 4. The most favorable locations for O2 addition are C6 and C5′, respectively. For CH3OO-β-CAR, O2 addition on C6 is the most favorable (-0.3 kcal/mol). In contrast, O2 addition on C5′ is the most favorable (-3.4 kcal/mol) for CH3OO-LYC. In the case of LAOO-β-CAR• and LAOO-LYC•, O2 additions on C6 are more favorable. Comparatively, ROO-β-CAR• is less likely to bind O2 than LAOO-LYC•. Nevertheless, O2 affinities of the ROO-CAR• adducts are significantly lower than those of carbon-centered radicals derived from lactones and LAH and are comparable to or smaller than those of β-CAR(-H)• and LYC(-H)• (see Table 1). The result is consistent with the conclusion that the formation of ROO-CAR-OO• is not favored unless under higher O2 pressure.1,13 Hydrogen abstractions of the ROO-CAR-OO• radicals (eq 9) are presented by the OO-H BDEs of ROO-CAR-OO-H. The computed values are similar to those of peroxyl OO-H BDEs. As summarized in Table 4, the OO-H BDEs are noticeably larger than the C-H BDEs of LAH, β-CAR, and LYC. For the CH3OO-CAR• + CH3OO• f CH3OO-CAROOCH3 reactions, the CH3OO• radical adds favorably on C6 and C5′. In contrast with the enthalpy of the addition of the first peroxyl radical (Table 2), the addition of the second peroxyl radical is considerably more exothermic. The additions of CH3OO• and LAOO• to the LYC systems are more exothermic

TABLE 4: Enthalpies of O2 Addition, OO-H BDE, CH3OO• Addition, And Hydrogen Addition (Negative of C-H BDE) of CH3OO-β-CAR• and CH3OO-LYC•b CAR

C6

C8

C10

C12

C14

C15′

C13′

C9′

C7′

12.9

10.8

10.9

10.9

9.4

8.7

76.7

CH3OO-CAR-OO-H f CH3OO-CAR-OO• + H• 76.1 76.0 76.2 74.6 73.4 74.7

74.0

74.4

75.9

76.0

74.9

74.1

74.9

CH3OO-CAR + O2 f CH3OO-CAR-OO 11.7 12.5 12.8 11.5 •

β-CAR LYC

β-CAR LYC

β-CAR LYC

β-CAR LYC a

4.6 (-0.3) 3.5 (-1.9)a 1.7 (-1.4) 1.4 (-2.4)a 75.6 (80.3) 74.9 (79.0)a 75.2 (80.6) 76.9 (81.4)a

9.3

11.0

9.1

10.5

11.3

75.4

11.9

75.2

10.3

74.8

9.8

74.0

C11′

C5′

•

4.1 (1.6) 4.6 (2.5)a 0.8 (-3.4) 1.8 (-2.3)a 74.7 (79.6) 74.7 (79.6)a 75.0 (79.9) 73.7 (78.3)a

-24.1 (-30.4) -22.7 (-29.6)a -28.6 (-32.7) -31.7 (-35.2)a

-24.4

CH3OO-CAR• + CH3OO• f CH3OO-CAR-OOCH3 -21.3 -20.7 -22.1 -19.9 -19.7 -19.9

-20.9

-25.2

-20.9

-20.0

-16.9

-17.3

-19.8

-27.3 (-30.4) -23.3 (-27.5)a -28.0 (-33.4) -29.1 (-35.1)a

-69.0 (-71.2) -69.8 (-72.7)a -66.3 (-68.7) -68.9 (-71.4)a

-59.8

-57.0

CH3OO-CAR• + H• f CH3OO-CAR-H -56.5 -55.9 -55.6 -55.1 -55.2

-56.2

-62.3

-60.9

-51.0

-57.2

-53.6

-56.7

-62.3

-18.5

-56.6

-17.8

-56.0

-17.2

-54.9

-16.7

-53.0

-55.4

CH3OO replaced with LAOO. b The lowest-energy (C5) CH3OO• and carotene adducts are considered. Data computed using MPWB1K/ 6-31++G(d,p)//B3LYP/6-31G(d) are in parentheses.



Figure 3. Reaction energy diagram from MPWB1K/6-31++G(d,p)//B3LYP/6-31G(d) for R ) CH3, L ) LA, and CAR ) β-CAR.

than to the β-CAR systems. Overall, the LYC system is energetically more favorable than β-CAR with respect to the consecutive scavenging of two peroxyl radicals. As shown in the last part of Table 4, enthalpies for hydrogen abstractions of ROO-CAR• (resulting in ROO-CAR-H) are similar for β-CAR and LYC and occur most favorably on C6. For LAOO-β-CAR• and LAOO-LYC•, enthalpies of hydrogen abstraction are -72.7 and -71.4 kcal/mol, respectively. ROO-βCAR• has a slightly higher tendency toward hydrogen abstraction than its ROO-LYC• counterparts. Referring to the C-H BDEs reported in Table 2, ROO-CAR• is capable of undergoing hydrogen abstraction from LAH, β-CAR, and LYC. It is noted, however, that the propensity for hydrogen abstraction of peroxyl radical is even stronger. The protective function of the addition adduct is thus exhibited by its significantly reduced O2 affinity and by its lower propensity toward hydrogen abstraction from the lipid than peroxyl radical. Reaction Diagrams. To compare both mechanisms, i.e., hydrogen abstraction and adduct formation, the reaction energy diagrams for the lipid model, peroxyl radical, and carotenes are illustrated in Figures 3-6. Figures 3 and 4 illustrate the reactions of CH3OO• with β-CAR and LYC, respectively. Figures 5 and 6 illustrate the reactions of LAOO• with β-CAR and LYC. Beginning with the ROO• radical, possible reactions include hydrogen abstraction from LAH, hydrogen abstraction from CAR, and addition to CAR. In all cases, it is observed that the addition reaction forming ROO-CAR• (eq 7) is the most exothermic. Hydrogen abstraction from CAR (eq 6), resulting in the formation of ROOH + CAR(-H)•, is slightly more favorable than hydrogen abstraction from the lipid. The O2 affinity of a radical is an essential factor that influences its tendency toward chain propagation. LA• binds significantly more strongly to O2 than CAR(-H)• and ROOCAR•. In contrast, ROO-CAR• has the smallest O2 affinity among these carbon-centered radicals. The adduct, being less reactive toward O2, prevents the chain propagation of lipid. The ROO-CAR• radical has a relatively small C-H BDE and thus is a good hydrogen donor (see Table 3). In the presence of a

second peroxyl radical, ROO-CAR• may undergo hydrogen donation (resulting in ROOH + ROO-CAR(-H, 5-4′), the numbers represent locations of addition and hydrogen abstraction, respectively) or addition (resulting in ROO-CAR-OOR). Both processes are favorable as shown at the lower-left of the figures. The formation of ROO-CAR• is thus a detour of ROO• for avoiding the otherwise deleterious effect of lipid peroxidation, i.e., the formation of ROOH + LAOO•. Comparatively, ROO-β-CAR• has a smaller O2 affinity than ROO-LYC•. ROO-LYC• is less likely to undergo hydrogen abstraction from the lipid than ROO-β-CAR•. This information suggests that under high oxygen pressure ROO-β-CAR• may prevent peroxidation to a larger extent than ROO-LYC•. In addition, under low oxygen pressure ROO-LYC• may provide better antioxidative protection than ROO-β-CAR•. LAOO represents a relatively more realistic model of lipid peroxide than CH3OO. The energy diagrams corresponding to R ) LA (Figures 5 and 6) are different from those corresponding to R ) CH3 (Figures 3 and 4) in several aspects. The formation of addition adducts by LAOO• is less exothermic than those of their CH3OO• counterparts. In addition, the relative energies of the species resulting from quenching of two LAOO• are less exothermic than those of their CH3OO• counterparts, as can be seen in the lower-left in Figures 3-6. Compared with CH3OO•, LAOO• is less likely to undergo hydrogen abstraction from LAH or CAR. The reaction diagrams are consistent with the experimental observation that at high oxygen pressures CAR loses its antioxidant activity or even becomes pro-oxidative.1,19 At a high O2 concentration, the equilibrium between ROO-CAR• + O2 and ROO-CAR-OO• shifts to the latter, which can undergo hydrogen abstraction from the lipid and propagate lipid peroxidation. Being less reactive toward O2 addition than CAR(-H)•, ROO-CAR• would react with a second ROO• to form either ROO-CAR-OOR (eq 10) or ROOH + ROO-CAR(-H) (eq 11). These are all very exothermic processes for both the β-CAR and the LYC systems. Similarly, β-CAR(-H)• and LYC(-H)•

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Figure 4. Reaction energy diagram from MPWB1K/6-31++G(d,p)//B3LYP/6-31G(d) for R ) CH3, L ) LA, and CAR ) LYC.

Figure 5. Reaction energy diagram from MPWB1K/6-31++G(d,p)//B3LYP/6-31G(d) for R ) L ) LA and CAR ) β-CAR.

react favorably with ROO• either by the formation of ROOCAR(-H,4-4′) (the numbers represent locations of hydrogen abstraction and addition, respectively) or by the second hydrogen abstraction on the carotenes. However, for the radical combinations to occur, the original radical has to be unreactive toward chain propagations. Therefore, the reaction path involving ROO-CAR• is more likely to offer antioxidative protection against lipid peroxidation. The addition mechanism first proposed by Burton and Ingold1 is supported by our computational data. The hydrogen abstraction product of CAR provides a certain degree of antioxidative function. The O2 affinities of CAR(-H)•

are larger than those of ROO-CAR•; however, they are noticeably smaller than that of LA•. In addition, C-H BDEs of CAR(-H)• are smaller than that of LAH. CAR(-H)• can scavenge the second peroxyl radical by either a second hydrogen abstraction to form CAR(-H2) or by addition with a peroxyl radical to from ROO-CAR(-H). The lowest energy addition products ware denoted as ROO-CAR(-H,4-4) in Figures 3-6, indicating that hydrogen abstraction and ROO• addition of CAR occur both on C4. These processes are very exothermic as shown in Figures 3-6. CAR(-H)-OO• and ROO-CAROO• undergo hydrogen abstractions from LAH or CAR readily as shown in the figures. At elevated concentrations of CAR, it



Figure 6. Reaction energy diagram from MPWB1K/6-31++G(d,p)//B3LYP/6-31G(d) for R ) L ) LA and CAR ) LYC.

has been proposed that ROO• and ROO-CAR• may undergo hydrogen abstraction from CAR and induce auto-oxidation.1 We chose two C40H56 carotenes, β-CAR and LYC, in our study. Supposedly these compounds function in the less polar region in the membranes, and thus the electron transfer mechanism is less important. Because of the relatively mediumsized models used in this study, our investigated systems are rather simplistic. In biological systems, the location and orientation of carotenoids within the membrane, medium polarity, and the property of various lipid peroxyl radicals are all intricate factors that would affect the functions of carotenoids.19 Our investigation, however simplified, provides the thermodynamic information related to the relevant species involved in the reactions of chain termination. We hope that our effort would provide valuable insight into future studies of antioxidant and pro-oxidant activities of carotenoids. 4. Conclusion The antioxidative, chain-breaking functions of carotenes via hydrogen abstraction and peroxyl radical addition have been examined using density functional theory. The addition mechanism, in which a peroxyl radical-carotene adduct (ROOCAR•) is formed at the onset of radical formation, was demonstrated to be thermodynamically favorable. The reaction energy diagrams obtained reveal that the addition mechanism provides a larger extent of antioxidative protection than hydrogen abstraction. The protective function of the addition adduct is demonstrated by its low O2 affinity and by its propensity to act as a hydrogen donor. The adducts are not likely to undergo propagations unless under high oxygen pressure. Being relatively stable, the adduct is able to quench the second peroxyl radical. Among the two investigated carotenes, ROO-βCAR• has a lower O2 affinity than ROO-LYC•. Through hydrogen abstraction, carotene species are also able to provide protection against lipid peroxidation, however to a lesser extent. Under low oxygen pressure, β-CAR(-H)• and LYC(-H)• may quench the second peroxyl radical via either radical combination

or second hydrogen abstraction. For the latter, the formation of LYC(-H2) is particularly energetically favorable. Acknowledgment. The authors acknowledge that the National Science Council of Taiwan, Republic of China, supported this work. We also thank the National Center for HighPerformance Computing for computer time and facilities. Supporting Information Available: Comparisons of selected BDEs computed using various density functionals from those obtained from G3 theory, C-H BDEs, and O2 affinities of carotenes and linoleic acid at different locations, detailed reaction enthalpies of species, and reaction diagrams obtained using B3LYP/6-31G(d). This material is available free of charge via the Internet at http://pubs.acs.org. References and Notes (1) Burton, G. W.; Ingold, K. U. Science 1984, 224, 569–573. (2) Edge, R.; McGarvey, D. J.; Truscott, T. G. J. Photochem. Photobiol. B: Biol. 1997, 41, 189–200. (3) Martin, H. D.; Ruck, C.; Schmidt, M.; Sell, S.; Beutner, S.; Mayer, B.; Walsh, R. Pure Appl. Chem. 1999, 71, 2253–2262. (4) Sies, H.; Stahl, W. Am. J. Clin. Nutr. 1995, 62 (suppl), 1315S– 1321S. (5) Stahl, W.; Sies, H. Arch. Biochem. Biophys. 1996, 336, 1–9. (6) Di Mascio, P.; Murphy, M. E.; Sies, H. Am. J. Clin. Nutr. 1991, 53 (suppl), 194S–200S. (7) Polidori, M. C.; Stahl, W.; Eichler, O.; Niestroj, I.; Sies, H. Free Radical Biol. Med. 2001, 30, 456–462. (8) Miller, N. J.; Sampson, J.; Candeias, L. P.; Bramley, P. M.; RiceEvans, C. A. FEBS Lett. 1996, 384, 240–242. (9) Truscott, T. G. J. Photochem. Photobiol. B: Biol. 1996, 35, 233– 235. (10) Yamauchi, R.; Kato, K. J. Agric. Food Chem. 1998, 46, 5066– 5071. (11) Burton, G. W. J. Nutr. 1989, 119, 109–111. (12) Anna, I.; Cristina, R.; Stefania, B.; Aldo, T.; Louise, M. C. J. Biochem. Mol. Toxic. 1998, 12, 299–304. (13) Kennedy, T. A.; Liebler, D. C. J. Biol. Chem. 1992, 267, 4658– 4663. (14) Mayne, S. T. FASEB J. 1996, 10, 690–701.

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JP907822H

Chain-Breaking Activity of Carotenes in Lipid Peroxidation: A

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