Kinetic Study of the Aroxyl-Radical-Scavenging Activity of Five Fatty

Article pubs.acs.org/JPCB

Kinetic Study of the Aroxyl-Radical-Scavenging Activity of Five Fatty Acid Esters and Six Carotenoids in Toluene Solution: Structure− Activity Relationship for the Hydrogen Abstraction Reaction Kazuo Mukai,*,† Maya Yoshimoto,† Masaharu Ishikura,‡ and Shin-ichi Nagaoka† †

Department of Chemistry, Faculty of Science, Ehime University, Matsuyama 790-8577, Japan Green Project, Business Development Center, Showa Denko K.K., Kawasaki 210-0858, Japan

‡

S Supporting Information *

ABSTRACT: A kinetic study of the reaction between an aroxyl radical (ArO•) and fatty acid esters (LHs 1−5, ethyl stearate 1, ethyl oleate 2, ethyl linoleate 3, ethyl linolenate 4, and ethyl arachidonate 5) has been undertaken. The secondorder rate constants (ks) for the reaction of ArO• with LHs 1− 5 in toluene at 25.0 °C have been determined spectrophotometrically. The ks values obtained increased in the order of LH 1 < 2 < 3 < 4 < 5, that is, with increasing the number of double bonds included in LHs 1−5. The ks value for LH 5 was 2.93 × 10−3 M−1 s−1. From the result, it has been clarified that the reaction of ArO• with LHs 1−5 was explained by an allylic hydrogen abstraction reaction. A similar kinetic study was performed for the reaction of ArO• with six carotenoids (Car-Hs 1−6, astaxanthin 1, β-carotene 2, lycopene 3, capsanthin 4, zeaxanthin 5, and lutein 6). The ks values obtained increased in the order of Car-H 1 < 2 < 3 < 4 < 5 < 6. The ks value for Car-H 6 was 8.4 × 10−4 M−1 s−1. The ks values obtained for Car-Hs 1−6 are in the same order as that of the values for LHs 1−5. The results of detailed analyses of the ks values for the above reaction indicated that the reaction was also explained by an allylic hydrogen abstraction reaction. Furthermore, the structure−activity relationship for the reaction was discussed by taking the result of density functional theory calculation reported by Martinez and Barbosa into account.

1. INTRODUCTION Carotenoids (Car-Hs) are localized in biomembranes and play an important role as antioxidants. Car-Hs show high singletoxygen-quenching activities.1−4 The second-order rate constant (kQ) for the reaction of Car-Hs with singlet oxygen (1O2) reported is ∼1010 M−1 s−1 (reaction 1). The values are about 2 orders of magnitude larger than those reported for natural phenolic antioxidants, including α-tocopherol (α-TocH), ubiquinol-10, and flavonoids (such as catechins and flavone derivatives).5 Measurements of the kQ values were also performed for vegetable and fruit extracts, including high concentration of Car-Hs, to investigate the 1O2 quenching activity of foods and plants.6,7

rived peroxyl radicals) in organic solvents, liposomes, liver microsomes, and cells in culture.10,11,17 For interaction of Car-Hs with ROO•, the following three reactions were considered: (i) electron transfer (reaction 2),9 (ii) radical addition (i.e., adduct formation) (reaction 3),8 and (iii) hydrogen atom transfer (i.e., allylic hydrogen atom abstraction) (reaction 4),13,16 forming the radical cation, adducts, and Car-H neutral radicals, respectively. Possible mechanisms for reactions 2−4 of Car-Hs with ROO• were discussed in the reviews by Edge and Truscott,18 Krinsky and Yeum,19 and El-Agamy et al.20

kQ

carotenoid + 1O2 → carotenoid (triplet excited state) 3

+ O2

(2)

ROO• + Car‐H → [ROO‐Car‐H]•

(3)

ROO• + Car‐H → ROOH + Car•

(4) 14−17

Kinetic studies were performed for reactions 2 and 3. However, measurements of the reaction rate for hydrogen abstraction reaction 4 of ROO• with Car-Hs have not been performed because the rates of reactions 2 and 3 are faster than

(1)

Reactions of Car-Hs with free radicals (ROO•, such as CCl3O2•, RSO2•, NO2•, aryl peroxyl radicals, and alkyl peroxyl radicals) were also studied by many investigators.8−17 Many studies have shown that Car-Hs exhibit protective effects against lipid peroxidation mediated by free radicals (lipid peroxyl radicals or 2,2′-azobis(2,4-dimethylvaleronitrile)-de© 2017 American Chemical Society

ROO• + Car‐H → ROO− + Car‐H+•

Received: May 12, 2017 Revised: June 27, 2017 Published: July 24, 2017 7593

DOI: 10.1021/acs.jpcb.7b04570 J. Phys. Chem. B 2017, 121, 7593−7601

Article

The Journal of Physical Chemistry B

(ksCar‑H) for Car-Hs 1−6, the structure−activity relationship for the hydrogen abstraction reaction in fatty acids and Car-Hs has been discussed.

those of reaction 4 and reactions 2 and 3 proceed before reaction 4 occurs. In previous studies, measurements of the second-order rate constant (kp) for the reaction of the 5,7-di-isopropyltocopheroxyl (5,7-Di-i-Pr-Toc•) radical with five fatty acid esters (LHs 1−5) (Figure 1) were performed in benzene

ks LH

ArO• + LH ⎯⎯⎯→ ArOH + L•

(7)

ksCar ‐ H

ArO• + Car‐H ⎯⎯⎯⎯⎯→ ArOH + Car •

(8)

2. EXPERIMENTAL METHODS 2.1. Materials. Five kinds of ethyl esters of fatty acids (LHs 1−5, ethyl stearate 1, ethyl oleate 2, ethyl linoleate 3, ethyl linolenate 4, and ethyl arachidonate 5 (>99%, respectively)) were purchased from Sigma Chemical Co. (St. Louis, MO) and used as received. Car-Hs (1−6) are commercially available. Astaxanthin 1 (Ast 1) was obtained from Funakoshi Co. Ltd., Japan. β-Carotene 2 (β-Car 2) and lycopene 3 (Lyc 3) were obtained from Wako Chemicals, Japan. Capsanthin 4 (Cap 4), zeaxanthin 5 (Zea 5), and lutein 6 (Lut 6) were obtained from Extrasynthese (Genay, France). ArO• was synthesized according to the method reported in a previous paper.28 2.2. Methods. The kinetic data were obtained on a Shimadzu spectrophotometer model UV-2100S by mixing equal volumes of toluene solutions of ArO• and LHs 1−5 (or Car-Hs 1−6) under a nitrogen atmosphere.21,22 All of the measurements were performed at 25.0 ± 0.5 °C. 3. RESULTS 3.1. Second-Order Rate Constants (ksLH) for the Reaction of Fatty Acid Ethyl Esters 1−5 with the Aroxyl Radical in Toluene. ArO• is stable in the absence of LHs 1−5 and shows absorption peaks at λmax = 377, 527, and 573 nm in toluene solution (Figure 3a and Table 2).29 On the other hand, LHs 1−5 show no absorption peak in the visible region. By adding a toluene solution of excess LHs 2−5 to a toluene solution of ArO•, the absorption spectrum of the ArO• gradually disappears. Figure 3b shows an example of the result of interaction between ArO• (8.85 × 10−5 M) and ethyl linoleate (LH 3) [(a) 1.83 × 10−2 M, (b) 3.66 × 10−2 M, (c) 5.49 × 10−2 M, and (d) 7.32 × 10−2 M] in toluene, showing that the decay rate of ArO• increases with increasing concentration of LH 3. The pseudo-first-order rate constant (kobsd) was obtained by varying the concentration of LH 3. The ArO• shows slow natural decay in toluene solution. Therefore, the pseudo-first-order rate constant (kobsd) for ArO• bleaching is given by eq 9.

Figure 1. Molecular structures of five LHs (1−5) (ethyl stearate 1, ethyl oleate 2, ethyl linoleate 3, ethyl linolenate 4, and ethyl arachidonate 5), α-tocopherol, and α-Toc•, 5,7-Di-i-Pr-Toc•, and aroxyl (ArO•) radicals. The positions of A- and B-type H-atoms (○ and □) activated by two π-electron systems and a single π-electron system, respectively, are shown in the LH 1−5 molecules.

(reaction 5).21,22 A similar measurement was performed for the reaction of the α-tocopheroxyl radical (α-Toc•) with LHs 1−5 in toluene (reaction 6).23,24 From the results obtained, it has been clarified that the reactions of 5,7-Di-i-Pr-Toc• and α-Toc• with LHs 1−5 may be explained by allylic hydrogen abstraction reactions.

−d[ArO• ]/dt = kobsd LH[ArO• ]

kp

5, 7‐Di‐i‐Pr‐Toc• + LH → 5, 7‐Di‐i‐Pr‐Toc‐H + L•

•

= {ko ArO + ks LH[LH]}[ArO• ]

(5) kp

α ‐Toc• + LH → α ‐Toc‐H + L•

(9)

•

where koArO is the rate constant for natural decay of ArO• in the medium and ksLH is the second-order rate constant for the reaction of ArO• with the added LH. These parameters are obtained by plotting kobsd against [LH], as shown in Figure 3c. The rate constant obtained for LH 3, ksLH, is (12.5 ± 0.1) × 10−4 M−1 s−1. Similar measurements were performed for the reaction of ArO• with unsaturated LHs (2, 4, and 5) in toluene solution (see Figures S2−S4, Supporting Information). On the other hand, in the case of ethyl stearate (LH 1) containing no double bond, the reaction between ArO• and LH 1 was negligibly slow (Figure S1). The ksLH values obtained are summarized in Table

(6)

In the present work, kinetic studies of ArO• (2,6-di-t-butyl-4(4′-methoxyphenyl)phenoxyl, Figure 1)-scavenging activity of five LHs 1−5 and six Car-Hs (Car-Hs 1−6, Figure 2) were performed in toluene solution, using a UV−visible spectrophotometer.21,22 A stable ArO• was used as a model of active free radicals, such as LOO•, LO•, and Toc•, as described in previous works.25−27 The second-order rate constants (ks) for the reaction of ArO• with five LHs 1−5 and six Car-Hs 1−6 (reactions 7 and 8) were measured in toluene solution. By comparing the ksLH values obtained for LHs 1−5 with those 7594


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Figure 2. Molecular structures of Car-Hs 1−6. The positions of A- and B-type H-atoms (○ and □) activated by two π-electron systems and a single π-electron system, respectively, are shown in the Car-Hs 1−6 molecules.

(d) 6.63 × 10−4 M)] in toluene. Under these conditions, the rate of disappearance of β-Car 2 in the presence of a constant concentration of ArO• is accurately first-order in β-Car 2. Therefore, the pseudo-first-order rate constant (kobsdβ‑Car) for βCar 2 bleaching is given by eqs 10-A,10-B.

1. As listed in Table 1, the rate constants (ksLH) increase as the number of double bonds in LHs 1−5 increases. 3.2. Second-Order Rate Constants (ksCar‑H) for the Reaction of Car-Hs 1−6 with the Aroxyl Radical in Toluene. Measurements of UV−vis absorption spectra of CarHs 1−6 were performed in toluene solution. The values of the absorption peak (λmax) and molar extinction coefficient (ε) obtained are listed in Table 2. Car-Hs generally show absorption peaks in the visible region, and the absorptions overlap with those of ArO• (Figure 4a). The values of molar extinction coefficient (ε) are about 1 order of magnitude larger than those of ArO• (see Table 2), as reported in previous studies.5,6 For example, β-Car 2 (Car-H 2) shows absorption peaks at λmax1 = 464 nm (ε1 = 103 000 M−1 cm−1) and λmax2 = 493 nm (ε2 = 89 000 M−1 cm−1) in toluene solution (Figure 4a). Therefore, it is difficult to determine the ksCar‑H values for the reaction of Car-Hs 1−6 with ArO• under the pseudo-firstorder condition ([ArO•] ≪ [Car-H]), by analyzing the decay curve of the ArO• at 377 and/or 573 nm, as performed for the reaction between ArO• and LHs 1−5. ArO• shows a weak absorption minimum at λmin = 430 nm (εmin = 167 M−1 cm−1) (see Figure 4a). On the other hand, the ε430 value of β-Car at 430 nm is 64 700 M−1 cm−1 in toluene. The εmin value of ArO• at 430 nm is 387 times smaller than the ε430 value of β-Car at 430 nm. Therefore, the measurement of the rate constant (ksCar‑H) for the reaction of ArO• with Car-Hs 1−6 was performed by reacting Car-Hs 1−6 with excess ArO•. The rate was measured by following the decrease in absorbance at 430 nm of Car-Hs 1−6 and by varying the concentration of ArO•. Figure 4b shows an example of the result of interaction between β-Car ([β-Car] = 2.48 × 10−5 M) and ArO• ([ArO•] = (a) 1.66 × 10−4 M, (b) 3.32 × 10−4 M, (c) 4.98 × 10−4 M,

−d[Car‐H]/dt = kobsd Car ‐ H[Car‐H] = {koCar ‐ H + ksCar ‐ H[ArO• ]}[Car‐H] (10-A)

kobsd

Car ‐ H

= ko

Car ‐ H

+ ks

Car ‐ H

•

[ArO ]

(10-B)

koCar‑H

where is the rate constant for natural decay of Car-Hs in the medium and ksCar‑H is the second-order rate constant for the reaction of ArO• with added Car-Hs. The ksCar‑H values are obtained by plotting kobsdCar‑H against [ArO•], as shown in Figure 4c. The ksCar‑H value obtained for β-Car 2 is (2.2 ± 0.4) × 10−4 M−1 s−1. Similar measurements were performed for the reaction of ArO• with Car-Hs 1−6 in toluene (see Figures S6−S10, Supporting Information). The ksCar‑H values obtained are summarized in Table 3. In the case of Ast 1, the reaction rate is very slow and we could not determine the rate constant (ksAst 1). As listed in Table 3, the rate constants (ksCar‑H) increase in the order of Car-H 1 < 2 < 3 < 4 < 5 < 6. The ksCar‑H values obtained for Car-Hs 2−6 are in the same order as that of those for LHs 2−5, as listed in Tables 1 and 3. The results of detailed analyses of the ksCar‑H values for the reaction of ArO• with Car-Hs 1−6 suggest that the reaction may be explained by the H-atom abstraction reaction (reaction 8). As shown in Figure 4, the reactions between ArO• and β-Car 2 were performed under the following condition: [ArO•] ≫ [βCar 2]. Therefore, if electron transfer and radical addition 7595


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reactions (reactions 2 and 3) occurred, UV−vis absorption of β-Car 2 (at λmax = 464 nm) would show a remarkable change. However, such a change was not observed, indicating that reactions 2 and 3 are not active for the interaction between ArO• and β-Car 2.

4. DISCUSSION 4.1. Structure−Activity Relationship in the AroxylRadical-Scavenging Reaction by Fatty Acid Ethyl Esters 1−5: Mechanism of the Reaction. First, measurements of the second-order rate constant (ksLH) for the reaction of ArO• with LHs 1−5 were performed in ethanol. However, we were unsuccessful in determining the rate constant because the reaction rates are very slow in ethanol. Generally, the ks values for the reaction of ArO• with antioxidants increase with decreasing polarity of solvent (i.e., dielectric constant (ε) of solvent).29 Therefore, in the present study, the ksLH values were measured in toluene solution having low polarity. As listed in Table 1, the ksLH values increased in the order of LH 1 < 2 < 3 < 4 < 5. The ksLH 1 value for LH 1 (i.e., saturated fatty acid ethyl ester 1) was negligible. By comparing the ksLH values obtained for ethyl oleate 2 and ethyl linoleate 3, it is observed that the value of the latter is ca. 4.4 times larger than that of the former. Ethyl oleate 2 has four −CH2− hydrogen (H)-atoms activated by a single π-electron system (−CHB2−CC−CHB2−) (hereafter, we call these −CHB2− H-atoms as B-type H-atoms) (see Figure 1). On the other hand, ethyl linoleate 3 has two −CH2− H-atoms activated by two π-electron systems (−CC−CHA2−C C−) (similarly, we call these −CHA2− H-atoms as A-type Hatoms) in addition to the above four B-type H-atoms. Consequently, these two A-type H-atoms will mainly contribute to the high reactivity of ethyl linoleate 3. As shown in Figure 1, the A- and B-type H-atoms in LHs 1−5 are marked as ○ and □, respectively. LHs 3, 4, and 5 have two, four, and six A-type H-atoms, respectively, and four B-type H-atoms. Therefore, generally, the ksLH values for LHs 2−5 may be calculated as the sum of the rate constants (ksA‑type + ksB‑type) due to the A- and B-type Hatoms included in LH 2−5 molecules (see eq 11). ks LH = ks A ‐ type + ks B ‐ type = m × kabstr A ‐ type + n × kabstr B ‐ type

(11)

where the ksA‑type and ksB‑type values are the products of the Hatom abstraction rate constants for A- and B-type H-atoms (kabstrA‑Type and kabstrB‑Type) and the numbers of A- and B-type H-atoms (m and n) included in LHs 2−5 molecules, respectively. Ethyl oleate 2 has only four B-type H-atoms (i.e., m = 0 and n = 4) and thus the rate constant, kabstrB‑type, given on an available hydrogen basis, is ksLH 2/4 (= ksB‑type/4) = 0.715 × 10−4 M−1 s−1 for LH 2. Ethyl linoleate 3 has two A-type Hatoms, in addition to the above four B-type H-atoms (i.e., m = 2 and n = 4) (see Figure 1). As the contribution of two A-type Hatoms (i.e., ksA‑type) is 12.5 × 10−4 − 4 × 0.715 × 10−4 M−1 s−1 = (12.5 − 2.86) × 10−4 M−1 s−1 (=9.64 × 10−4 M−1 s−1), the rate constant (kabstrA‑type), given on an available hydrogen basis, is estimated to be (9.64 × 10−4)/2 M−1 s−1 = 4.82 × 10−4 M−1 s−1 for LH 3. Similarly, the kabstrA‑type values can be calculated to be 4.68 × 10−4 and 4.41 × 10−4 M−1 s−1 for LHs 4 and 5, respectively, indicating good accordance with that (4.82 × 10−4

Figure 3. (a) UV−visible absorption spectrum of the aroxyl radical (ArO•) in toluene. (b) Decay of ArO• for the reaction of ArO• with ethyl linoleate 3 observed at 376 nm in toluene at 25.0 °C. (c) Dependence of the pseudo-first-order rate constant (kobsdLH 3) on the concentration of ethyl linoleate 3 in toluene. 7596


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Table 1. Rate Constants (ks (ArO•)) for the Reaction of Five Fatty Acid Ethyl Esters 1−5 with the Aroxyl Radical (ArO•) in Toluene at 25.0 °C, Number of A- and B-type H-atoms (m and n), ksA‑type Values, and kabstrA‑type and kabstrB‑type Values (See the Text) ksLH/M−1 s−1

lipid ethyl ethyl ethyl ethyl ethyl a

stearate 1 oleate 2 linoleate 3 linolenate 4 arachidonate 5

slow (2.86 (12.5 (21.6 (29.3

± ± ± ±

0.11) × 10−4a 0.1) × 10−4 0.2) × 10−4 0.3) ×10−4

number of A-type H-atoms (m)

number of B-type H-atoms (n)

0 2 4 6

4 4 4 4

kabstrA‑type and kabstrB‑type/M−1 s−1

ksA‑type/M−1 s−1

−4

−4

(12.5 − 2.86) × 10 = 9.64 × 10 (21.6 − 2.86) × 10−4 = 18.74 × 10−4 (29.3 − 2.86) × 10−4 = 26.44 × 10−4

0.715 × 10−4 (B-type) 4.82 × 10−4 (A-type) 4.68 × 10−4 (A-type) 4.41 × 10−4 (A-type)

Errors are standard deviations (SD).

Table 2. UV−Vis Absorption Maxima (λmaxi, i = 1−3) and Molar Extinction Coefficients (εi, i = 1−3) of Car-Hs 1−6 and the Aroxyl Radical in Toluene Solution Car-H Ast 1 β-Car 2 Lyc 3 Cap 4 Zea 5 Lut 6 aroxyl radical

λmax1/nm (ε1/M−1 cm−1) 486 464 458 483 463 432 377

(112 000) (103 000) (97 000) (103 000) (114 000) (80 000) (11 800)

λmax2/nm (ε2/M−1 cm−1)

λmax3/nm (ε3/M−1 cm−1)

493 (89 000) 485 (143 000)

520 (123 000)

492 (99 000) 457 (118 000) 527 (2400)

486 (106 000) 573 (2700)

by the H-atom abstraction reactions between the radicals and LHs 2−5. 4.2. Comparison between the Aroxyl-Scavenging Rate Constants (ksCar‑H) and Hydrogen Atom Dissociation Energy (ΔE) of Car-Hs 1−6. In the present study, the second-order-rate constants (ksCar‑H) for the reaction of ArO• with Car-Hs 1−6 were determined in toluene solution. The rate constants (ksCar‑H) increased in the order of Car-H 1 < 2 < 3 < 4 < 5 < 6, as listed in Table 3. As discussed in Section 4.1, the reaction of ArO• with LHs 1−5 was well-explained by the H-atom abstraction mechanism. Both A- and B-type H-atoms activated by double and single π-bond systems in LHs 1−5, respectively, contributed to the ArO•-scavenging activity, that is, ArO•-scavenging rate constant (ksLH). As shown in Figure 2, the A- and B-type H-atoms (○ and □) are contained in CarHs 1−6 studied, suggesting that the reaction between ArO• and Car-Hs 1−6 may also be explained by the H-atom abstraction mechanism. Molecular orbital calculations were used to estimate the freeradical- and reactive-oxygen-scavenging activities of CarHs.34−36 Density functional theory (DFT) calculations (BPW91/D5DV) were performed for 13 Car-Hs (see Figure 1 in ref 34) by Martinez and Barbosa,34 to evaluate the contribution of the H-atom abstraction mechanism (reaction 4) in Car-Hs. Four Car-Hs (Ast 1, β-Car 2, Zea 5, and Lut 6) used in the present study are included in their calculation. As the structure of the left half of Cap 4 is the same as that of Zea 5, the values calculated for Zea 5 were used for Cap 4. The calculation of binding energy (ΔE/eV) for 4-oxo-rubixanthin having the structure similar to that of Lyc 3 is also included. ΔE values estimated for A- and B-type H-atoms in Car-Hs 1−6, using the ΔE values calculated by Martinez and Barbosa (see Figures 3 and 4 in ref 34), are listed in Table 3. The ΔE value obtained for B-type H-atoms activated by a single π-electron system (−CC−CHB2−R) is 3.1 eV for βCar 2, Cap 4, Zea 5, and Lut 6, showing their same value. As DFT calculation is not performed for Lyc 3, the value (3.0 eV) calculated for 4-oxo-rubixanthin (see Figure 1 in ref 34) was used for Lyc 3, showing good accordance with the above value (3.1 eV). The value (3.2 eV) for B-type H-atoms activated by the −CC−CHB(OH)−R system in Lut 6 is also similar to that (3.0 and 3.1 eV) for Car-Hs 2−5. On the other hand, the ΔE value obtained for A-type H-atoms activated by two CC double bonds (CC−CHA2−CC) is 2.7 eV for Lut 6, as shown in Figures 3 and 4 in ref 34. The value is much smaller than those (3.0, 3.1, and 3.2 eV) for B-type H-atoms in Car-Hs 2−5, as expected.21 The ΔE values obtained for H-atoms of the CH3-group activated by a single π-electron system (−CC−CH3) are 3.5

M−1 s−1) obtained for LH 3. The kabstrA‑type values are ca. 6.7− 6.2 times larger than the kabstrB‑type value. As reported in previous studies, the reaction rate constants (kp) for the H-atom abstraction reaction of LHs (1−5) by 5,7Di-i-Pr-Toc• and α-Toc• were measured using a usual UV−vis spectrophotometer21,22 and double-mixing stopped-flow spectrophotometer,24 respectively. The reactions of α-Toc• with LHs 2−5 are well known as a pro-oxidant reaction of α-TocH in edible oils.30−33 For example, the second-order rate constants (kp) obtained for the reaction with α-Toc• are very small and negligible for LH 1, 1.90 × 10−2 M−1 s−1 for LH 2, 8.33 × 10−2 M−1 s−1 for LH 3, 1.92 × 10−1 M−1 s−1 for LH 4, and 2.43 × 10−1 M−1 s−1 for LH 5 in toluene at 25.0 °C.24 Although the absolute values of the rate constants (ks) obtained for the reaction of LHs 2, 3, 4, and 5 with ArO• (see Table 1) are ca. 66, 67, 89, and 83 times smaller than those obtained with α-Toc• in toluene, respectively, the relative rate constants obtained for the former are similar to those for the latter. A similar tendency was obtained for the reaction of LHs 1−5 with 5,7-Di-i-PrToc•.21,22 To interpret the results obtained, ab initio molecular orbital calculations of models for LHs 1−5 were carried out.21 The C− H bond dissociation energy (D) decreased, and the C−H bond length (r) increased in the models of LHs 1−3 as the number of CC double bonds increased, and these were nearly constant in the models of LHs 3−5. From the calculation results, it was shown that the observed features of the rate constant could be explained in terms of the pseudo-πconjugation between the −CC− double bond and the active hydrogen−carbon bond of the −CH2− group bound to the −CC− double bond (−CH2−CC−CH2− or −CC− CH2−CC−). As described above, the reactions of ArO•, αToc•, and 5,7-Di-i-Pr-Toc• with LHs 2−5 were well-explained 7597


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eV for Ast 1, 3.4 eV for β-Car 2, 3.3 eV for Lyc 3, 3.2 eV for Cap 4 and Zea 5, and 3.2 and 3.9 eV for Lut 6 (see Figure 1 in ref 34). The ΔE values vary remarkably from 3.2 to 3.9 eV. Ast 1 has two B-type H-atoms activated by a single CO πelectron system (OC−CHB(OH)−) (Figure 2), and ΔE is 3.1 eV. However, as the ksAst 1 value of Ast 1 is small and negligible, the effect of CO double bonds for hydrogen atom abstraction in Ast 1 seems to be negligible. Similarly, the contribution of H-atom abstraction of the CH3-group activated by a single π-electron system (−CC−CH3) is also negligible for Ast 1 because of large ΔE value (3.5 eV). As β-Car 2 and Zea 5 have a similar π-electron system, we may expect similar ksCar‑H values (i.e., ArO•-scavenging activity) for β-Car 2 and Zea 5. However, the ks value of Zea 5 is larger than that of β-Car 2 (Table 3). The ΔE value (3.4 eV) calculated for H-atoms of the CH3-group activated by a single π-electron system (−CC−CH3) for β-Car 2 is larger than that (3.2 eV) for Zea 5. Consequently, the ksβ‑Car 2 value of βCar 2 will be smaller than that of Zea 5 because H-atoms of the CH3-group in β-Car 2 do not contribute to the hydrogen atom abstraction reaction. Furthermore, H-atoms of the CH3-group in Lyc 3 also do not contribute to the H-atom abstraction reaction because the ksLyc 3 value of Lyc 3 is smaller than that of Cap 4 and Zea 5. The results suggest that H-atoms of CH3groups having the ΔE value 3.2 eV contribute to H-atom abstraction. On the other hand, H-atoms of CH3-groups having a ΔE value larger than 3.3 eV do not contribute to H-atom abstraction. However, the detailed reason is not clear at present. The other C−H bonds included in Car-Hs 1−6 show larger ΔE values (see Figures 3 and 4 in ref 34), suggesting that the contribution of these C−H bonds to the hydrogen abstraction reaction of Car-Hs 1−6 is small and negligible. Formation of Car-H neutral radicals (Car•) in photosystem II was reported by Gao et al.37 based on the results of measurements of electron nuclear double resonance and visible/near-IR spectra and time-dependent DFT calculations. An absorption peak observed at 750 nm was assigned to the neutral radical with a proton loss from the 4(4′) position of the β-Car radical cation (see Chart 1 in ref 37). The result shows good agreement with those obtained by DFT calculation34 and by measurement of the reaction of ArO• with β-Car 2. As reaction 8 indicates, Car-H neutral radicals (Cars•) will be produced by the reaction between ArO• and Car-Hs. However, as Cars• are unstable in solution,37 we could not observe the absorption spectra of Cars• in toluene at 25 °C. Jeevarajan et al.38 reported UV−vis absorption spectra of the cation radical (Car+•) (λmax = 970 nm) and dication (Car2+) (λmax = 817 nm) of β-Car 2 produced by the reaction of β-Car 2 with ferric chloride in dichloromethane. As shown in Figure 4 in ref 38, broad absorptions of Car+• and Car2+ were observed in the ∼600−1200 and ∼500−1000 nm regions, respectively. Therefore, if Car+• and Car2+ coexist in toluene solution, we may observe absorption spectra of Car+• and Car2+. However, spectra due to Car+• and Car2+ were not observed for the reaction between ArO• and Car-Hs 1−6, including β-Car 2, as described in previous Section 3.2. Furthermore, formation of a neutral Car-H radical (Car•) from a Car-H radical cation (Car+•) and dication (Car2+) was reported by Kispert et al.39,40 If these Car•, Car+•, and Car2+ and their reaction products coexist in toluene solution and the absorptions of these compounds overlap with those of Car-Hs 1−6, the decay of Car-Hs 1−6 at 430 nm due to the reaction with ArO• does not follow the pseudo-first-order reaction. As the results of the

Figure 4. (a) UV−visible absorption spectrum of ArO• and β-Car 2 in toluene. (b) Decay of β-Car 2 for the reaction with ArO• observed at 430 nm in toluene at 25.0 °C. (c) Dependence of the pseudo-firstorder rate constant (kobsdβ‑Car 2) on the concentration of ArO• in toluene. 7598


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Table 3. Rate Constants (ks (ArO•)) for the Reaction of Car-Hs 1−6 with Aroxyl Radical (ArO•) in Toluene at 25.0 °C, Number of A- and B-Type H-Atoms (m and n), kabstrA‑type and kabstrB‑type Values, and Binding Energy (ΔE) (See the Text) Car-H

a

ks

Car‑H

/M

−1

s

−1

number of A-type H-atoms (m)

number of B-type H-atoms (n)

kabstr

A‑type

and

kabstrB‑type/M−1

s

−1

Ast 1 β-Car 2 Lyc 3

slow (2.2 ± 0.4) × 10−4a (2.4 ± 0.5) × 10−4

0 0 0

0 4 4+4=8

0.55 × 10−4 (B-type) 0.30 × 10−4 (B-type)

Cap 4 Zea 5

(2.8 ± 0.9) × 10−4 (3.1 ± 0.4) × 10−4

0 0

2+3=5 4 + 6 = 10

0.56 × 10−4 (B-type) 0.31 × 10−4 (B-type)

Lut 6

(8.4 ± 0.3) × 10−4

1

2+1+3=6

(8.40−0.55 × 6) × 10−4 = 5.1 × 10−4 (A-type)

binding energy (ΔE/eV) and number of A- and B-type H-atoms (m and n) 3.1 (n = 2 × 2 = 4, CH2) 3.0 (n = 2 × 2 = 4, CH2), 3.0 (n = 2 × 2 = 4, CH2) 3.1 (n = 2, CH2), 3.2 (n = 3, CH3) 3.1 (n = 2 × 2 = 4, CH2), 3.2 (n = 3 × 2 = 6, CH3) 2.7 (m = 1, CH), 3.1 (n = 2, CH2), 3.2 (n = 1, CH2), 3.2 (n = 3, CH3)

Errors are (SD).

kabstrA‑type value) was estimated to be (8.4 − 6 × 0.55) × 10−4 M−1 s−1 (= 5.1 × 10−4 M−1 s−1). The kabstrA‑type value is 9.3 times larger than the kabstrB‑type value and shows reasonable agreement with those [(4.41−4.82) × 10−4 M−1 s−1] obtained for LHs 3, 4, and 5. Similarly, using eq 12 and numbers, n, of 8, 5, and 10 for CarHs 3, 4, and 5, kabstrB‑type values were determined to be 0.30 × 10−4, 0.56 × 10−4, and 0.31 × 10−4 M−1 s−1, respectively. These values show considerable accordance with that (0.55 × 10−4 M−1 s−1) obtained for β-Car 2. Furthermore, these values also show reasonable agreement with that (0.715 × 10−4 M−1 s−1) obtained for ethyl oleate 2. These results indicate that the ArO•-scavenging rates obtained for Car-Hs 1−6 are due to the hydrogen abstraction reaction from A- and B-type H-atoms included in Car-H molecules. Contribution of the other C−H bonds included in Car-Hs 1−6 is considered to be small and negligible. The second-order rate constants (ksLH) for the reaction of LHs 1−5 with ArO• were well-explained by a hydrogen abstraction mechanism, as described in Section 4.1. Similarly, ksCar‑H values obtained for the reaction of Car-Hs 1−6 with ArO• were also explained by a mechanism of simple hydrogen abstraction reaction (reaction 4). Contributions of the electron transfer mechanism (reaction 2) and radical addition mechanism (reaction 3) seem to be negligible, differing from the reactions of free radicals (such as CCl3O2•, RSO2•, NO2•, aryl peroxyl radicals, and alkyl peroxyl radicals) with CarHs.8−17 The ksCar‑H values (2.2 × 10−4 − 3.1 × 10−4 M−1 s−1) obtained for Car-Hs 2−5 are similar to those (2.86 × 10−4 M−1 s−1) for ethyl oleate 2 and smaller than those (12.5 × 10−4 M−1 s−1) for ethyl linoleate 3. On the other hand, the ksα‑TocH value reported for α-TocH is 9.87 × 104 M−1 s−1 in benzene.29 The ksCar‑H values obtained for Car-Hs 2−6 (2.2 × 10−4 − 8.4 × 10−4 M−1 s−1) are about 8 orders of magnitude smaller than those for α-TocH in benzene. Aroxyl-type free-radicalscavenging activities of Car-Hs are very low compared to those for α-TocH.

analysis of decay reaction of Car-Hs 2−6 (Figures 4 and S6− S10, Supporting Information) show, the decay of Car-Hs 2−6 was well-explained by a simple pseudo-first-order reaction. The result also suggests that electron transfer and ArO•-addition reactions do not occur between ArO• and Car-Hs 1−6 and that only hydrogen abstraction reaction 8 contributes to the reaction, as observed for reaction 7 between ArO• and LHs 2−5. However, detailed mechanisms are not clear at present. 4.3. Structure−Activity Relationship in the AroxylRadical-Scavenging Reaction by Car-Hs 1−6: Mechanism of the Reaction. As discussed in Section 4.2, the reaction of ArO• with Car-Hs 1−6 may be explained by the Hatom abstraction reaction mechanism. The results of DFT calculation suggest that A- and B-type H-atoms contained in Car-Hs 1−6 contribute to the reaction of Car-Hs. The A- and B-type H-atoms having small binding energies (ΔE) are marked as ○ and □, respectively, in Figure 2. By comparing the rate constants (ksCar‑H) obtained for Lut 6 and β-Car 2, is observed that the ksLut 6 value of the former is ca. 3.8 times larger than the ksβ‑Car 2 value of the latter (see Table 3). β-Car 2 has four B-type H-atoms activated by a single πelectron system (−CC−CHB2−) (see Figure 2). On the other hand, Lut 6 has one A-type H-atom activated by two πelectron systems (−CC−CHAR−CC−) in addition to three B-type H-atoms activated by a single π-electron system. Consequently, this one A-type H-atom will mainly contribute to the high reactivity of Lut 6, judging from the results obtained for LHs 1−5. Furthermore, Car-Hs 1, 2, 3, 4, and 5 have only n = 0, 4, 8, 5, and 10 B-type H-atoms, respectively. It seems that the ksCar‑H value increases with an increase in the number of Btype H-atoms (n), as listed in Table 3. Therefore, as performed for LHs 1−5, the ksCar‑H values for Car-Hs 1−6 may also be calculated as the sum of the rate constants (ksA‑type + ksB‑type) due to the A- and B-type H-atoms included in Car-Hs 1−6 (eq 12). ksCar ‐ H = ks A ‐ type + ks B ‐ type = m × kabstr A ‐ type + n × kabstr B ‐ type

(12)

5. CONCLUSIONS Measurements of the second-order rate constants (ks) for the reactions of five LHs (1−5) and six Car-Hs (1−6) with ArO• were undertaken in toluene at 25 °C to clarify the mechanism and structure−activity relationship of the reactions. Rate constants (ks) of Car-Hs 1−6 were similar to those of ethyl oleate 2 and 8 orders of magnitude smaller than those of αTocH in benzene. Although Car-Hs generally show very high

β-Car 2 has only B-type four H-atoms (i.e., m = 0 and n = 4) and thus the rate constant (kabstrB‑type) given on an available hydrogen basis is estimated to be (2.2 × 10−4)/4 M−1 s−1 = 0.55 × 10−4 M−1 s−1. Lut 6 has one A-type H-atom, in addition to the above six Btype H-atoms (i.e., m = 1 and n = 6) (see Figure 2). Therefore, using eq 12 and the value of kabstrB‑type obtained for β-Car 2, contribution of one A-type H-atom in Lut 6 (i.e., ksA‑type = 7599


Article

The Journal of Physical Chemistry B activity for quenching of 1O2, the result of the present kinetic study indicates that the aroxyl-type free-radical-scavenging activity of Car-Hs is very low. The reactions of ArO• with LHs 1−5 and Car-Hs 1−6 were well-explained by the allylic Hatom abstraction reaction. Both A- and B-type H-atoms activated by double and single π-bond systems, respectively, in LHs 1−5 (and Car-Hs 1−6) and their numbers (m and n) contributed to the ArO•-scavenging rate constant (ks), that is, the ArO•-scavenging activity (see eqs 11 and 12).

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for carotenoids and food extracts. J. Agric. Food Chem. 2011, 59, 3717− 3729. (7) Iwasaki, Y.; Takahashi, S.; Aizawa, K.; Mukai, K. Development of singlet oxygen absorption capacity (SOAC) assay method. 4. Measurements of the SOAC values for vegetable and fruit extracts. Biosci., Biotechnol., Biochem. 2015, 79, 280−291. (8) Burton, G. W.; Ingold, K. U. β-Carotene: An unusual type of lipid antioxidant. Science 1984, 224, 569−573. (9) Grant, J. L.; Kramer, V. J.; Ding, R.; Kispert, L. D. Carotenoid cation radicals: Elecrochemical, optical, and EPR study. J. Am. Chem. Soc. 1988, 110, 2151−2157. (10) Terao, J. Antioxidant activity of β-carotene-related carotenoids in solution. Lipids 1989, 24, 659−661. (11) Lim, B. P.; Nagao, A.; Terao, J.; Tanaka, K.; Suzuki, T.; Takama, K. Antioxidant activity of xanthophylls on peroxyl radical-mediated phospholipid peroxidation. Biochim. Biophys. Acta, Lipids Lipid Metab. 1992, 1126, 178−184. (12) Liebler, D. C. Antioxidant reactions of carotenoids. Ann. N. Y. Acad. Sci. 1993, 691, 20−31. (13) Liebler, D. C.; McClure, T. D. Antioxidant reactions of βcarotene: Identification of carotenoid-radical adducts. Chem. Res. Toxicol. 1996, 9, 8−11. (14) Mortensen, A.; Skibsted, L. H. Importance of carotenoid structure in radical-scavenging reactions. J. Agric. Food Chem. 1997, 45, 2970−2977. (15) Mortensen, A.; Skibsted, L. H.; Sampson, J.; Rice-Evans, C.; Everett, S. A. Comparative mechanisms and rates of free radical scavenging by carotenoid antioxidants. FEBS Lett. 1997, 418, 91−97. (16) Woodall, A. A.; Lee, S. W.-M.; Weesie, R. J.; Jackson, M. J.; Britton, G. Oxidation of carotenoids by free radicals: relationship between structure and reactivity. Biochim. Biophys. Acta, Gen. Subj. 1997, 1336, 33−42. (17) Woodall, A. A.; Britton, G.; Jackson, M. J. Carotenoids and protection of phospholipids in solution or in liposomes against oxidation by peroxyl radicals: Relationship between carotenoid structure and protective ability. Biochim. Biophys. Acta, Gen. Subj. 1997, 1336, 575−586 and references are cited therein.. (18) Edge, R.; Truscott, T. G. Carotenoid Radicals and the Interaction of Carotenoids with Active Oxygen Species. In The Photochemistry of Carotenoids; Frank, H. A., Young, A. J., Britton, G., Cogdell, R. J., Eds.; Kluwer Academic Publishers: Netherlands, 1999; Chapter 12, pp 223−234. (19) Krinsky, N. I.; Yeum, K.-J. Carotenoid-radical interactions. Biochem. Biophys. Res. Commun. 2003, 305, 754−760 and references are cited therein.. (20) El-Agamey, A.; Lowe, G. M.; McGarvey, D. J.; Mortensen, A.; Phillip, D. M.; Truscott, T. G.; Young, A. J. Carotenoid radical chemistry and antioxidant/pro-oxidant properties. Arch. Biochem. Biophys. 2004, 430, 37−48 and references are cited therein.. (21) Nagaoka, S.; Okauchi, Y.; Urano, S.; Nagashima, U.; Mukai, K. Kinetic and ab initio study of the prooxidant effect of vitamin E. Hydrogen abstraction from fatty acid esters and egg yolk lecithin. J. Am. Chem. Soc. 1990, 112, 8921−8924. (22) Mukai, K.; Morimoto, H.; Okauchi, Y.; Nagaoka, S. Kinetic study of reactions between tocopheroxyl radicals and fatty acids. Lipids 1993, 28, 753−756. (23) Remorova, A. A.; Roginsky, V. A. Rate constants for the reaction of α-tocopherol phenoxy radicals with unsaturated fatty acid esters, and the contribution of this reaction to the kinetics of inhibition of lipid oxidation. Kinet. Catal. 1991, 32, 726−731. (24) Ouchi, A.; Ishikura, M.; Konishi, K.; Nagaoka, S.; Mukai, K. Kinetic study of the prooxidant effect of α-tocopherol. Hydrogen abstraction from lipids by α-tocopheoxyl radical. Lipids 2009, 44, 935− 943 and references are cited therein.. (25) Mukai, K.; Kageyama, Y.; Ishida, T.; Fukuda, K. Synthesis and kinetic study of antioxidant activity of new tocopherol (vitamin E) compounds. J. Org. Chem. 1989, 54, 552−556. (26) Mukai, K.; Tokunaga, A.; Itoh, S.; Kanesaki, Y.; Ohara, K.; Nagaoka, S.; Abe, K. Structure-activity relationship of the free-radical-

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jpcb.7b04570. Figures S1−S4: (a) Decay of ArO• for the reaction of ArO• with four LHs (1, 2, 4, and 5) (ethyl stearate 1, ethyl oleate 2, ethyl linolenate 4, and ethyl arachidonate 5), respectively, observed at 376 nm in toluene at 25.0 °C; (b) dependence of the pseudo-first-order rate constant (kobsdLH) on the concentration of LH in toluene; Figure S5−S9: (a) UV−visible absorption spectrum of ArO• and five Car-Hs (Car-Hs 1 and 3− 6) (Ast 1, Lyc 3, Cap 4, Zea 5, and Lut 6), respectively, in toluene; (b) decay of Car-H for the reaction with ArO• observed at 430 nm in toluene at 25.0 °C; (c) dependence of the pseudo-first-order rate constant (kobsdCar‑H) on the concentration of ArO• in toluene (PDF)

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: 81-89-927-9590. ORCID

Kazuo Mukai: 0000-0003-4118-9607 Shin-ichi Nagaoka: 0000-0003-1564-7328 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We are very grateful to Dr. Aya Ouchi of Ehime University for her kind help in the measurement of the reaction rates. This work was partly supported by JSPS KAKENHI Grant Number 15k07431.

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REFERENCES

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Kinetic Study of the Aroxyl-Radical-Scavenging Activity of Five Fatty

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