Article pubs.acs.org/JPCB
β‑Carotene As a Lipophilic Scavenger of Nitric Oxide Rui-Min Han,† Hong Cheng,† Ruopei Feng,† Dan-Dan Li,† Wenzhen Lai,*,† Jian-Ping Zhang,† and Leif H. Skibsted*,‡ †
Department of Chemistry, Renmin University of China, Beijing, 100872, China Food Chemistry, Department of Food Science, University of Copenhagen, Rolighedsvej 30, DK-1058 Frederiksberg C, Denmark
‡
ABSTRACT: The efficient bleaching following continuous bubbling of gaseous nitric oxide (NO•) to β-carotene (β-Car) dissolved in n-hexane under anaerobic conditions results from an initial addition of two NO• followed by fragmentation coupled with further NO• addition as shown by mass spectrometry (MS). Density functional theory (DFT) calculations demonstrated that hydrogen atom transfer (HAT) and electron transfer (ET) from β-Car to NO• are strongly energetically unfavorable in contrast to radical adduct formation (RAF) followed by degradation. The results indicated the lowest energy for addition of the first NO• at C7 with an activation free energy of ΔG≠ = 74.40 kJ mol−1 and a rate constant of 0.56 s−1, followed by trans-addition of a second NO• at C8 with ΔG≠ = 55.51 kJ mol−1. MS confirmed the formation of a dinitrosyl-β-Car (596.6 m/z), and of a β-Car fragment (400.4 m/z) formed by C7/C8 bond cleavage and suggested to be of importance for progression of bleaching. Up to eight reaction products with increasing mass of 28 m/z are assigned to continuous addition of NO• to the initially formed fragment forming nitroxides. Continuous wave photolysis of sodium nitroprusside (SNP) as a NO• source dissolved together with β-Car in 4:1 (v/v) methanol:tetrahydrofuran gradually bleached β-Car. Nanosecond laser flash photolysis at 355 nm followed by transient absorption spectroscopy showed a β-Car derived intermediate with an absorption maximum around 420 nm in agreement with a prediction (425 nm) from time-dependent DFT (TDDFT) for the trans-C7,8 dinitrosyl adduct of β-Car. The NO• adduct of βCar decays with a rate constant of ∼107 s−1 at 25 °C.
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INTRODUCTION Carotenoids affect oxidative processes in biological systems by the quenching of excited states and by the scavenging of radicals.1−6 Carotenoids are lipophilic, and their protective effects are linked to membranes often in synergistic interaction with hydrophilic plant phenols.7,8 As for radical scavenging, radical adduct formation (RAF) competes with electron transfer (ET) and hydrogen atom transfer (HAT).9−13 HAT for β-carotene (β-Car) is only known for the strongly oxidizing hydroxyl radical •OH, while more reducing radicals like the superoxide radical anion O2•− may even be oxidized by some of the least reducing carotenoids like astaxanthin (Ast)4,9 •
•
β‐Car + OH → β‐Car( −H) + H 2O
(1)
Ast + O2•− → Ast•− + O2
(2)
not directly available for scavenging by the phenols or phenolates.14 Carotenoids were recently shown to be optimally regenerated by moderately reducing plant phenolates in agreement with the Marcus theory for electron transfer.8 Nitric oxide (NO•) is involved in numerous biological functions including aqueous phase antioxidant activities.15−20 NO• is a relatively long-lived radical and may be reducing or oxidizing depending on the actual conditions15,16,19
(3)
NO• + H+ + e− → HNO
(5)
Received: July 28, 2014 Revised: September 14, 2014 Published: September 16, 2014
Such regeneration of β-Car results in antioxidant synergism since regenerated β-Car may continue to scavenge lipid radicals © 2014 American Chemical Society
(4)
NO• is in the aqueous phase of mammalian muscles bound to heme pigments like myoglobin,18 which serves as an NO• reservoir. However, NO• is also soluble in lipids and may easily diffuse from one phase to another phase with different polarity. The effect of NO• interaction with lipophilic antioxidants like carotenoids on the overall antioxidant efficiency remains unknown.21 NO• is, however, known to add to β-Car forming nitroxides.22,23 Nitroxides are stable radicals and as such potential antioxidants.
The interaction of β-Car and other carotenoids with hydrophilic antioxidant occurs at lipid/water interfaces, where less efficient water-soluble antioxidants (φ-OH) mainly in the deprotonated form may regenerate β-Car from the β-Car radical cation (β-Car•+) as the oxidation product from scavenging of lipid radicals in the lipid phase2,7 β‐Car •+ + φ‐O− → β‐Car + φ‐O•
NO• + H 2O → NO2− + 2H+ + e−
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calculated using time-dependent DFT (TDDFT) method. The effect of solvent, n-hexane, was included by using the integralequation-formalism polarizable continuum model (IEFPCM)27 in the single-point calculations and TDDFT calculations. After testing six functionals (BPW91, B3PW91, M06-2X, PBE0, CAMB3LYP, and wB97XD), wB97XD functional was chosen, since it predicts the absorption at 439 nm for β-Car and 1015 nm for β-Car radical cation, in good agreement with experimental results.9,28 The geometry optimizations were preformed in the gas-phase with the wB97XD functional and 631G* basis set (labeled as B1). Each stationary point was classified as a minimum or transition state by the frequency calculations. Then the energy was corrected by using a larger basis set, 6-311+G(2d,2p), labeled as B2. The free energies could then be calculated from the B2 single-point electronic energy with addition of Gibbs free energy correction from the frequency calculation and the energy difference between gas and solution phase at the B1 level.
For the complicated pattern of antioxidant interactions at water/lipid interfaces as in membranes, knowledge of NO• reaction with carotenoids is missing. Such interaction may be important for function and integrity of membranes. We have accordingly studied the initial step in the reaction of NO• with β-Car combining experimental methods with theoretical calculations in order to establish stoichiometry of such reactions and to identify effects on both NO• as a bioregulator and on functions of β-Car as a membrane stabilizing antioxidant.24
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MATERIALS AND METHODS Chemicals. All-trans-β-carotene (β-Car) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). NO• (99.95%) was purchased from Yanglilai Chemical Gas Ltd. (Beijing, China). Sodium hydroxide and sodium nitroprusside (SNP, ≥96.0%) were purchased from Beijing Chemical Works (Beijing, China). Anhydrous calcium chloride (≥96.0%) was purchased from Aoboxing Biotechnology Co., Ltd. Beijing, China. HPLC-grade tetrahydrofuran (THF, Mreda Technology Inc., USA), n-hexane (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China), and methanol (Siyou Fine Chemical Co. Ltd. Tianjin, China) were used as received. Reaction of β-Carotene with Gaseous NO•. NO• gas was purified by bubbling through a NaOH (5 M) aqueous solution and dried by anhydrous CaCl2 to remove NO2 and water, respectively, before bubbling into a 2.0 × 10−6 M β-Car n-hexane solution under anaerobic conditions. Steady state UV−vis absorption spectra during the reaction of β-Car with gaseous NO• were recorded in a 1 cm optical cell on a Cary50 spectrophotometer (Agilent Technologies Inc., Santa Clara, CA). Mass spectrometry (LC/MS) of the final product in nhexane was determined using Bruker Apex IV FTMS (Irvine, CA). Reaction of β-Carotene with NO• Formed Photochemically from Sodium Nitroprusside. Binary solvent methanol:THF = 4:1 (v/v) was used to solubilize both lipophilic β-Car (4.0 × 10−5 M) and hydrophilic SNP (1.2 × 10−2 M). Samples in a 1 cm optical cell were exposed to light from a broad spectrum Xe lamp under anaerobic condition for increasing exposure time. β-Car (4.0 × 10−5 M) and SNP (1.2 × 10−2 M) each in methanol:THF = 4:1 (v/v) solution were photolyzed separately and when combined in solution. Laser Flash Photolysis. The laser flash photolysis apparatus has been described elsewhere.9 Briefly, the excitation laser pulses at 355 nm (3−5 mJ/pulse, 7 ns, 10 Hz) were supplied by a Nd3+:YAG laser (Quanta-Ray PRO-230; Spectra Physics, Santa Clara, CA), and the probe light provided by a xenon-flash lamp synchronized to the electric gate of the ICCD detector (L7685, Hamamatsu Photonics, Hamamatsu City, Japan). Solutions of samples were 1.0 × 10−4 M β-Car and 1.2 × 10−2 M SNP and photolyzed immediately after preparation. The optical path length of the flow cuvette (∼20 mL) used for laser flash photolysis was 5 mm. The anaerobic condition was achieved by bubbling the solution with high-purity argon for ∼30 min. All of the measurements were carried out at room temperature (25 °C). Computational Details. The reaction of β-Car and NO• radical was investigated using density functional theory (DFT) with the Gaussian 09 program package.25 All-trans conformation of β-Car was used since it was found to be the most stable isomer by a theoretical study.26 The absorption energies and intensities of the products in all the studied channels were
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RESULTS Nitric oxide (NO•) is known to bleach β-Car, and an excess of NO • corresponding to a NO • /β-Car molar ratio of approximately 10 was for tetrachloromethane as an electron withdrawing solvent found to result in complete bleaching of the visible absorption.22 The bleaching of β-Car indicates that the polyene conjugation in β-Car is disrupted. For n-hexane as solvent, NO• was likewise found to bleach β-Car when added in excess, see Figure 1.
Figure 1. Spectral changes during reaction of β-Car (4.0 × 10−6 M) with NO• in n-hexane at 25 °C for continuing bubbling of gaseous NO• into β-Car solution.The initial curve: the absorption spectrum of β-Car in n-hexane.
The stoichiometry of the reaction of NO• with β-Car became clear from a mass spectrometric analysis of the reaction mixtures, see Figure 2. One major reaction product was found to have a mass of 596.6 corresponding to β-Car with two NO• added (Scheme 1). This compound together with a minor reaction product with the higher mass of 624.6 and many degradation products of lower mass helped to track the main reaction path for the apparent rather complex degradation of βCar exposed to excess of NO•. The reaction of β-Car with NO• was further studied under conditions where NO• was generated in situ photochemically from SNP dissolved in 4:1 methanol:THF under anaerobic conditions,29 using continuous wave photolysis with light from a broad spectrum Xe lamp, and using laser flash photolysis 11660
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Figure 2. ESI mass spectra of the product of β-Car reaction with excess of NO• in n-hexane.
Scheme 1. Mechanism of Initial Reaction of β-Car in nHexane Involving Two NO•
Figure 3. UV−visible absorption spectra of (a) SNP (1.2 × 10−2 M), (b) β-Car (4.0 × 10−5 M), and (c) SNP (1.2 × 10−2 M) in the presence of β-Car (4.0 × 10−5 M) in methanol:THF = 4:1 (v/v) binary solvent as determined using 1 cm optical cell. Samples exposed to broad spectrum Xe lamp under anaerobic condition for increasing exposure time.
Fe(CN)5NO3− and β-Car dissolved, each at the same concentration as for the separate photolysis experiment, clearly showed an increasing bleaching of β-Car (Figure 3c). β-Car accordingly are bleached not only by direct light exposure, a well-known process,31 but also through reaction with a photoproduct of Fe(CN)5NO3− identified as NO• on the basis of spectral characteristics of the other photoproduct, Fe(CN)5(solvent)3−, cf. eq 6. β-Car is almost depleted completely within 6 min. The increasing absorbance at 400 nm for SNP (Figure 4a), corresponding to disappearance of Fe(CN)5NO3− as the result of light irradiation, shows similar dependence on exposure time in the absence of β-Car as in the presence of β-Car except for minor absorption increase at 4−6 min in the presence of β-Car. β-Car does accordingly not interfere with the photochemical formation of NO• from Fe(CN)5NO3−. The product of β-Car reacting with NO• formed from photolysis of Fe(CN)5NO3− decays completely within 10 min. The absorption spectrum of the product formed from reaction of β-Car with NO• may thus be obtained as the difference in absorption spectra between SNP and SNP/β-Car solutions at 6 min irradiation. The product spectrum shows absorption between 350−500 nm as seen in Figure 6 (blue line). In contrast, the rate of absorbance decrease at 480 nm, corresponding to the reaction of β-Car following light exposure, clearly depends on the presence of Fe(CN)5NO3− (Figure 4b). The presence of Fe(CN)5NO3− in the photolysis solution accordingly strongly accelerates the reaction of β-Car adding
(monochromatic at 355 nm in 7 ns pulses) combined with transient absorption spectroscopy for a dynamic characterization. Both methods used the photochemical reaction of eq 6 for a controlled NO• release to facilitate identification of the initial reaction products formed prior to the subsequent degradation reactions: Fe(CN)5 (NO)3 − + solvent → [Fe(CN)5 (solvent)]3 − + NO•
(6)
The steady-state absorption spectra of a solution of SNP (1.2 × 10−2 M) are shown in Figure 3a for increasing Xe lamp irradiation time. The primary photolysis reaction of Fe(CN)5NO3− corresponds to the reaction of eq 6, and the increasing absorption at λmax = 402 nm is similar to what has been observed for aqueous solutions.29,30 β-Car is also sensitive to light from the Xe lamp, and the absorbance intensity of βCar solution (4.0 × 10−5 M) is shown to decrease for increasing irradiation time (Figure 3b). Photolysis of solutions with both 11661
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Figure 6. Red line: difference absorption spectra between SNP/β-Car and SNP in methanol:THF = 4:1 (v/v) binary solvent at 30−50 ns in Figure 5. Black line: absorption spectra at 60 s from Figure 1 of β-Car (4.0 × 10−6 M) bubbled with NO•. Blue line: difference absorption spectra between SNP (1.2 × 10−2 M) and SNP (1.2 × 10−2 M)/β-Car (4.0 × 10−5 M) in methanol:THF = 4:1 (v/v) binary solvent at 6 min exposed to broad spectrum Xe lamp in Figure 3a,c. Green line: absorption spectra of trans-C7,8-β-Car···(NO)2 obtained from DFT theoretical calculation.
Figure 4. Time evolution profilesas obtained from Figure 3 for (a) SNP and SNP in the presence of β-Car at 400 nm, and (b) β-Car and β-Car in the presence of SNP at 480 nm.
further support to the role of the photochemically formed NO• as reagent for β-Car in a reaction parallel to the direct photolysis of β-Car. Laser flash photolysis with nanosecond pulses at 355 nm mainly results in excitation of Fe(CN)5NO3− as may be concluded from the relative absorbance of β-Car and Fe(CN)5NO3− (Figure 3a,b). A transient absorption at 370− 500 nm has a lifetime in the nanosecond time regime (Figure 5a,b). The difference spectrum with maximum at 420 nm (red
Three types of reactions, electron transfer (ET), hydrogen atom transfer (HAT), and radical adduct formation (RAF), which all previously were considered for the reaction of β-Car with the hydroxyl radical •OH, will also be considered for the reaction between NO• and β-Car, as they all will affect the conjugated polyene backbones of β-Car: ET:
β‐Car + NO• → β‐Car•+ + NO−
(7)
HAT:
β‐Car + NO• → β‐Car(− H)• + HNO
(8)
RAF:
β‐Car + NO• → (β‐Car···NO)•
(9)
For the ET reaction, experimental standard reduction potentials for β-Car•+/Car (E° = 1.06 V)11 and NO•, H+/ HNO (E = −0.55 V)15 combined with pKa = 7.2 for HNO,16 indicated that the ET reaction of eq 7 is not possible with ΔG° = 196 kJ mol−1 (Table 1), while the reduction of β-Car•+ by NO− is spontaneous. The ET reaction will accordingly not be considered further in this study. To gain more insight into the reaction mechanism, DFT calculations were performed for the HAT and RAF reactions. We consider only the reactivity of one-half of β-Car, up to C15 (Scheme 1) due to its symmetry. For the HAT reactions, the hydrogen atoms connected to C1a, C2, C3, C4, C5a, C9a, and C13a were considered. For the RAF reactions, the following reaction sites at β-Car were considered: C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, and C15. The calculated absolute energy (in a.u.) for the products formed by the reaction of βCar with one NO• at the B1 level is listed in Table 2, showing that the products for the HAT reactions have much higher energies than that for the RAF reactions. This is in agreement with the larger reaction free energy (ΔG° = 238 kJ mol−1) estimated from BDE for hydrogen abstraction from both β-Car and HNO (Table 1). Therefore, further calculations were accordingly only performed for the RAF reactions. For the RAF reactions, we have optimized the geometries of the reactant complexes, transition states, and product complexes for all of the possible addition paths. The calculated activation free energy and the reaction free energy for addition one NO• to β-Car are given in Table 3. As shown in Table 3, the addition reactions to C5 has the lowest reaction barrier ΔG≠, while the additions to C7 and C9 have reaction barriers close to that of C5 (within 0.5 kJ•mol−1). However, the
Figure 5. Transient absorption spectra at (a) 30−50 ns and (b) 100− 400 ns for SNP (1.2 × 10−2 M) in the presence and absence of β-Car (1.0 × 10−4 M) in methanol:THF = 4:1 (v/v) binary solvent following 355 nm photoexcitation with 7 ns pulse.
line, Figure 6) between the transient spectra of Fe(CN)5NO3− in the absence of β-Car and in the presence of β-Car is tentatively assigned to the absorbance of a transient species formed from β-Car reacting with NO•. The lifetime of this transient species with an absorption maximum at 420 nm is around 100 ns as it decays exponentially with a rate constant k ≈ 107 s−1 at 25 °C. The spectral characteristics of the two species, photochemical intermediate product of β-Car and nitroprusside (blue line in Figure 6) and intermediate product at 60 s (Figure 1) of excess NO• with β-Car (black line in Figure 6) are very similar. 11662
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Table 1. Reaction Free Energies (ΔG°, kJ mol−1) for ET, HAT, and RAF Reaction of β-Car with NO• and •OH ΔG° (kJ mol−1) ET HAT RAF
•
•
reaction mechanism
R = NO
R• = •OH
β-Car + R• → β-Car•+ + R− β-Car + R• → β-Car(−H)• + HR β-Car + R• → (β-Car···R)• (β-Car···R)• + R• → (β-Car···R2)
196a 238c 22(C7)e −11(trans-C7, 8)g
−69b −214d −142(C5)f
a Calculated from the standard reduction potentials for β-Car•+ and NO•, and pKa of HNO from refs 11, 15, and 16. bFrom ref 12. cCalculated from bond dissociation energy (BDE) as reported in refs 10 and 15. dFrom ref 9. eNO• addition to C7 of β-Car; fFrom ref 13. •OH addition to C5. gA second NO• addition to C7.
Table 4. Calculated Acitvation Free Energy (ΔG≠ in kJ mol−1) and Reaction Free Energy (ΔG° in kJ mol−1) of Second NO• RAF Paths after the First NO• Addition on the C7 Site, the Maximum Wavelength (λmax in nm) and Oscillator Strength (f) of the Absorption Spectra of the Corresponding Products
Table 2. Calculated Absolute Energy (in a.u.) for the Products Formed from the Reaction of β-Car with One NO• Molecule at the B1 Level RAF
HAT
site
E(B1)
site
E(B1)
C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15
−1687.229026 −1687.188445 −1687.232015 −1687.204954 −1687.223974 −1687.210503 −1687.221507 −1687.214865 −1687.220180 −1687.218331 −1687.220251
C1a C2 C3 C4 C5a C9a C13a
−1687.142918 −1687.147445 −1687.152866 −1687.194530 −1687.186858 −1687.184895 −1687.182531
Table 3. Calculated Activation Free Energy (ΔG≠ in kJ mol−1), Reaction Free Energy (ΔG° in kJ mol−1) for RAF Paths of First NO• Addition to β-Car, and the Maximum Wavelength (λmax in nm) and Oscillator Strength (f) of the Absorption Spectra of the Corresponding Products
a
site
ΔG≠a
ΔG°a
λmax
F
C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15
74.36 160.30 74.40 91.08 74.74 99.57 81.05 92.59 77.92 79.17 76.37
44.48 151.69 21.95 95.01 52.58 86.53 56.76 72.86 63.29 53.30 63.66
494 435 471 423 458 407 434 381 423 360 394
4.07 4.15 3.83 3.76 3.42 2.48 3.09 1.99 2.40 1.82 0.96
sitea
ΔG≠
ΔG°
λmax
f
C8 trans-C8 C10 trans-C10 C12 trans-C12 C14 trans-C14 C7′ C9′ trans-C9′ C11′ trans-C11′ C13′ trans-13′ C15′ trans-C15′
56.46 55.51 57.93 50.55 58.46 51.60 49.99 55.29 54.02 48.18 41.94 53.26 54.69 57.32 59.71 55.95 48.35
−3.77 −10.60 14.23 3.61 9.72 10.65 2.18 7.50 7.00 8.23 8.01 10.43 15.06 16.84 12.36 21.08 6.48
423 425 406 406 385 386 361 359 403 385 392 372 372 361 372 342 347
3.64 3.78 3.34 3.31 2.99 2.98 2.80 2.78 3.81 3.45 3.42 3.15 3.14 2.67 3.14 3.06 2.83
a The prefix “trans-” was used when the second NO• addition occurred in the different direction of the β-Car plane compared with the first NO• addition.
for the trans-addition, suggesting that both additions can be expected to occur. From an inspection of Table 5, it may be concluded that the second NO• addition after the initial addition to the C5 site is endothermic, which reveals that adduct formed by first NO• addition to C5 site is unlikely to be observed by experiments. The formation of dinitrosyl-β-Car accordingly follows the pattern outlined in Scheme 1 leading to the trans-7,8-dinitrosyl-β-Car as the isomer favored. The mononitrosyl-β-Car will be an intermediate of fleeting existence not building up to any significant concentration in homogeneous solution. The energy profile for NO• addition to β-Car is displayed in Scheme 2. This scheme also includes the initial step forming the reaction complexes for the addition of the first and the second NO• prior to the activation step. The reaction begins with the formation of the reactant complex [βCar](NO•), which is calculated to be 26.56 kJ•mol−1 less stable than free β-Car and NO•. The first NO• addition to C7 was favored with a reaction free energy ΔG° = 21.95 kJ•mol−1, an activation free energy ΔG≠=74.40 kJ•mol−1, and a rate constant k=0.56 s−1 calculated using conventional transition state theory (TST).4 The second NO• addition reaction starts with formation of the [β-Car-7-NO•](NO•) adduct and occurs
Including solvent effect (n-hexane).
reaction free energy clearly indicates that C7 should be the preferred reaction site from a thermodynamic point of view. Due to the radical character of mononitrosyl-β-Car, it can further react with the NO radical. Accordingly, we also investigated the second NO• addition reactions after the initial addition at the C5 and C7 sites. From the results summarized in Tables 4 and 5, it can be seen that the reactions for the second NO• additions have lower reaction free energies and barriers compared with that for the first NO• additions. As shown in Table 4, most of the second addition processes after the initial addition to C7 site were found to be endothermic. The exceptions are the cis- and trans-addition to the C8 site. The barrier for the cis-addition is only 1 kJ mol−1 higher than 11663
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initially seen both in the reaction between NO• and β-Car and in the photochemical reaction between Fe(CN)5NO3− and βCar. The reaction of NO• with β-Car and other carotenoids may have significant impact in vivo. The first NO• addition leads to delocalization of the single electron from NO• along the conjugated polyene backbone, which may relate to antioxidant activity of β-carotene toward NO•. The dinitrosyl adduct of βCar seems to have some stability as a nonradical and may carry NO• in lipophilic parts of membranes. The further reaction of β-Car with NO• will rather lead to nonreversible fragmentation for larger excess of NO•. The fragmentation pattern of β-Car seen for large excess of NO• in n-hexane indicates increments in molecular weight between fragments of 28 m/z, which could be explained by formation of stable nitroxide radicals from a fragment formed by cleavage between C7 and C8 in the initially formed C7/C8 dinitrosyl adduct (Scheme 3). As seen from the mass spectrum of the reaction mixture of βCar and NO•, the fragment peaks with m/z values ranging from 456.4 to 624.8 are evenly separated with increments of 28 m/z between each two adjacent peaks. This group of fragment peaks can be assigned as follows. The conjugated chain of β-Car undergoes cleavage of the double bond C7C8, and the larger fragment has a molecular weight of 400 m/z, which is in agreement with the fragment peak seen in the mass spectrum with a m/z value at 400.4. The addition of NO• to the β-Car fragment may be initiated by hydrogen atom abstraction as shown in Scheme 3. The net increase in molecular weight of the fragments seen in Scheme 3 is 28 m/z, which is in agreement with the interval of the m/z values of the fragment peaks from 456.4 to 624.8 of the mass spectrum. The stable nitroxide radical may further react with more NO• in the presence of excess NO•. This should give an explanation for the mass spectrum of the radical addition products of β-Car and NO•, while the variance in relative abundances of each fragment peak may be related to the stoichiometry of the reactions between β-Car and NO•. The initial mononitrosyl adduct of the fragment with a molecular weight of 428 m/z is not seen in the mass spectrum, but the fragment peaks with m/z values ranging from 456.4 to 624.8 may be assigned to the adduct products of 2 to 8 NO• to the 400 m/z fragment (Scheme 3).
Table 5. Calculated Activation Free Energy (ΔG≠ in kJ mol−1) and Reaction Free Energy (ΔG° in kJ mol−1) of Second NO• RAF Paths after the First NO• Addition on the C5 Site, the Maximum Wavelength (λmax in nm) and Oscillator Strength (f) of the Absorption Spectra of the Corresponding Products sitea
ΔG≠
ΔG°
λmax
f
C8 trans-C8 C10 trans-C10 C12 trans-C12 C14 trans-C14 C7′ trans-C7′ C9′ trans-C9′ C11′ trans-C11′ C13′ trans-13′ C15′ trans-C15′
53.76 54.95 60.03 52.19 59.97 50.63 50.45 62.52 57.60 50.10 53.89 55.05 48.28 51.44 58.24 49.82 48.47 57.17
3.78 11.43 18.94 11.84 23.64 5.46 9.50 19.13 4.27 11.40 14.87 25.35 14.20 17.85 31.94 21.89 5.92 17.28
425 421 405 406 387 386 363 363 482 418 408 410 394 397 381 385 365 364
3.76 3.77 3.36 3.43 3.11 3.11 3.20 3.15 2.97 3.91 3.53 3.60 3.13 3.19 2.71 2.83 2.74 2.77
The prefix “trans-” was used when the second NO• addition occurred in the different direction of the β-Car plane compared with the first NO• addition. a
through trans-addition at C8 with ΔG° = −10.60 kJ•mol−1 and ΔG≠ = 55.51 kJ•mol−1. β‐Car + 2NO• ⇄ β‐Car(7,8‐(NO)2 )
(10)
•
The concentration of NO in n-hexane is higher than the concentration of β-Car and a significant concentration of βCar(7,8-(NO)2) will appear.32 The further reactions as are evident from the MS will remove the dinitrosyl product and force the reaction of eq 10 to the product side. The calculations further indicate an absorption maximum for the dinitrosyl addition product with λmax = 425 nm (green line, Figure 6). The spectra agree reasonably well with the absorption observed by transient absorption spectroscopy following laser flash photolysis of mixture of β-Car and SNP, and with the absorption observed both during the photochemical reaction of β-Car with SNP and during the reaction of excess NO• with β-Car (Figure 6). The theoretical calculations accordingly provide evidence for the nature of the reaction intermediate
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DISCUSSION Nitric oxide, NO•, is a radical which like the hydroxyl radical, • OH, has high molecular mobility. The two radicals, however, function differently in biological systems due to significant differences in reactivity. •OH has been shown to react with β-
Scheme 2. Reaction Energy Diagram of β-Car with NO• Forming the C7,8 Adducta
a
Energies based on DFT-calculation are given in kJ mol−1. 11664
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Scheme 3. Suggested Mechanism for NO• Addition to β-Car Fragment Formed Following C7/C8 Carbon Bond Cleavage in n-Hexane during Reaction with Excess NO•a
mononitrosyl with a large negative ΔG° will favor C7 addition over C5 addition for NO•. It seems accordingly safe to conclude that the initial NO• addition to β-Car result in enhancement of reactivity of β-Car toward NO• and probably also other radicals. Such phenomena has previously been related to functions of carotenoids as molecular wires in membranes releasing oxidative stress from membrane surface to lipophilic antioxidants in membrane interior.24 The importance of the NO• interaction with β-Car is still speculative, but the facile stepwise addition of NO• to carotenoids deserves further attention. The initial addition of two NO• to β-Car is followed by degradation of β-Car through further reaction with NO• as suggested in Scheme 3. The initial two NO• addition reactions seem according to the theoretical calculation to be driven by the further degradation. The stricking consistency in a difference of 28 m/z between various reaction products resulting from the reaction between β-Car and excess NO• finds an explanation through addition of an increasing number of NO•. In contrast to the two initially added NO•, further addition seem to involve abstraction of hydrogen atoms, in agreement with the increasing mass of 28 m/z corresponding to addition of NO• and abstraction of two hydrogen atoms (14 + 16 − 2) (Scheme 3). The mechanism behind abstraction of hydrogen is not obvious, but NO• may disproportionate in the reaction mixture under the influence of catalytically active reaction intermediates:33 4NO• → N2 + 2NO2
(11)
NO2, as resulting from such disproportionation reactions, is known to abstract hydrogen atoms from β-Car.34,35 The nitroxide derivatives formed through reaction of NO• with βCar may serve as antioxidants in membranes being formed through diffusion of NO• into the lipophilic membrane for reaction with β-Car.
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CONCLUSIONS The reaction of NO• with β-Car seems to depend on the level of NO•. For reaction of β-Car with NO• for low levels of NO•, the reactivity of mononitrosyl adducts toward radicals is higher than for β-Car itself. For higher concentrations of NO•, β-Car may form lipophilic nitroxide derivatives with good antioxidant capabilities through further addition of NO•.
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a
AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected]. Tel: +86-10-6251-6604. Fax: +86-10-6251-6444. *E-mail: [email protected]. Tel: +45-3528-3221. Fax: +45-35283344.
Stereochemistry of intermediates and products are unknown.
Car through the HAT mechanism abstracting a hydrogen atom from β-Car with a large negative free energy of reaction resulting in formation of the neutral β-Car radical, β-Car(-H) • (Table 1).9 Other radicals including NO• will not react with βCar through a HAT reaction. For NO• and β-Car, ΔG° for HAT, as calculated from BDE for β-Car and HNO, is strongly positive, a result confirmed by the present DFT calculations. According to available thermodynamic data, NO• will not oxidize β-Car to β-Car•+.15 In contrast, NO• will react with βCar through RAF as is already known from experiments22,23 and now confirmed by DFT calculations. The ΔG° = 21.95 kJ mol−1 of Table 1 refers to addition of NO• to C7, while ΔG° = −142 kJ mol−1 refers to addition of •OH to C5 of β-Car. However, the subsequent addition of NO• to C8 in the
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work has been supported by the Fundamental Research Funds for the Central Universities, and the Research Fund of Renmin University of China (RUC No. 10XNI007, 14XNLQ04, 12XNLJ04, 14XNH058). LHS thanks the Danish Research Council for Independent Research, Technology and Production Science continuing support currently as the Grant 09-065906/FTP: Redox Communication in the digestive tract. 11665
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(22) Gabr, I.; Patel, R. P.; Symons, M. C. R.; Wilson, M. T. Novel Reactions of Nitric-Oxide in Biological-Systems. J. Chem. Soc. Chem. Commun. 1995, 915−916. (23) Mendiara, S. N.; Baquero, R. P.; Katunar, M. R.; Mansilla, A. Y.; Perissinotti, L. J. Reaction of beta-Carotene with Nitrite Anion in a Homogeneous Acid System. An Electron Paramagnetic Resonance and Ultraviolet-Visible Study. Appl. Magn. Reson. 2009, 35, 549−567. (24) Liang, J.; Tian, Y.-X.; Yang, F.; Zhang, J.-P.; Skibsted, L. H. Antioxidant Synergism Between Carotenoids in Membranes. Astaxanthin as a Radical Transfer Bridge. Food Chem. 2009, 115, 1437− 1442. (25) Frisch, M. J.; et al. Gaussian 09, revision C.01; Gaussian, Inc.: Wallingford, CT, 2009. (26) Cerón-Carrasco, J. P.; Bastida, A.; Zúñiga, J.; Requena, A.; Miguel, B. Density Functional Theory Study of the Stability and Vibrational Spectra of the β-Carotene Isomers. J. Phys. Chem. A 2009, 113, 9899−9907. (27) Cances, E.; Mennucci, B.; Tomasi, J. A New Integral Equation Formalism for the Polarizable Continnum Model: Theoretical Background and Applications to Isotropic and Anisotropic Dielectrics. J. Chem. Phys. 1997, 107, 3032−3041. (28) Tian, Y.-X.; Han, R.-M.; Zhang, J.-P.; Skibsted, L. H. Effect of Polar Solvents on β-Carotene Radical Precursor. Free Radic. Res. 2008, 42, 281−286. (29) de Oliveira, M. G.; Langley, G. J.; Rest, A. J. Photolysis of the [Fe(CN)5(NO)]2− Ion in Water and Poly(vinyl alcohol) Films: Evidence for Cyano Radical, Cyanide Ion and Nitric Oxide Loss and Redox Pathways. J. Chem. Soc., Dalton Trans. 1995, 2013−2019. (30) Shishido, S. M.; de Oliveira, M. G. Photosensitivity of Aqueous Sodium Nitroprusside Solutions: Nitric Oxide Release Versus Cyanide Toxicity. Prog. React. Kinet. Mec. 2001, 26, 239−261. (31) Nielsen, B. R.; Mortensen, A.; Jørgensen, K.; Skibsted, L. H. Singlet Versus Triplet Reactivity in Photodegradation of C-40 Carotenoids. J. Agric. Food Chem. 1996, 44, 2106−2113. (32) Shaw, A. W.; Vosper, A. J. Solubility of Nitric Oxide in Aqueous and Nonaqueous Solvents. J. Chem. Soc. Faraday Trans. 1977, 73, 1239−1244. (33) Nguyen, J. G.; Johnson, C. A.; Subramaniam, B.; Borovik, A. S. Nitric Oxide Disproportionation at Mild Temperatures by a Nanoparticulate Cobalt(II) Complex. Chem. Mater. 2008, 20, 5939− 5941. (34) Cerón-Carrasco, J. P.; Bastida, A.; Requena, A.; Zúñiga, J.; Miguel, B. A Theoretical Study of the Reaction of β-Carotene with the Nitrogen Dioxide Radical in Solution. J. Phys. Chem. B 2010, 114, 4366−4372. (35) Baker, D. L.; Krol, E. S.; Jacobsen, N.; Liebler, D. C. Reactions of β-Carotene with Cigarette Smoke Oxidants. Identification of Carotenoid Oxidation Products and Evaluation of the Prooxidant/ Antioxidant Effect. Chem. Res. Toxicol. 1999, 12, 535−543.
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β‑Carotene As a Lipophilic Scavenger of Nitric Oxide Rui-Min Han,† Hong Cheng,† Ruopei Feng,† Dan-Dan Li,† Wenzhen Lai,*,† Jian-Ping Zhang,† and Leif H. Skibsted*,‡ †
Department of Chemistry, Renmin University of China, Beijing, 100872, China Food Chemistry, Department of Food Science, University of Copenhagen, Rolighedsvej 30, DK-1058 Frederiksberg C, Denmark
‡
ABSTRACT: The efficient bleaching following continuous bubbling of gaseous nitric oxide (NO•) to β-carotene (β-Car) dissolved in n-hexane under anaerobic conditions results from an initial addition of two NO• followed by fragmentation coupled with further NO• addition as shown by mass spectrometry (MS). Density functional theory (DFT) calculations demonstrated that hydrogen atom transfer (HAT) and electron transfer (ET) from β-Car to NO• are strongly energetically unfavorable in contrast to radical adduct formation (RAF) followed by degradation. The results indicated the lowest energy for addition of the first NO• at C7 with an activation free energy of ΔG≠ = 74.40 kJ mol−1 and a rate constant of 0.56 s−1, followed by trans-addition of a second NO• at C8 with ΔG≠ = 55.51 kJ mol−1. MS confirmed the formation of a dinitrosyl-β-Car (596.6 m/z), and of a β-Car fragment (400.4 m/z) formed by C7/C8 bond cleavage and suggested to be of importance for progression of bleaching. Up to eight reaction products with increasing mass of 28 m/z are assigned to continuous addition of NO• to the initially formed fragment forming nitroxides. Continuous wave photolysis of sodium nitroprusside (SNP) as a NO• source dissolved together with β-Car in 4:1 (v/v) methanol:tetrahydrofuran gradually bleached β-Car. Nanosecond laser flash photolysis at 355 nm followed by transient absorption spectroscopy showed a β-Car derived intermediate with an absorption maximum around 420 nm in agreement with a prediction (425 nm) from time-dependent DFT (TDDFT) for the trans-C7,8 dinitrosyl adduct of β-Car. The NO• adduct of βCar decays with a rate constant of ∼107 s−1 at 25 °C.
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INTRODUCTION Carotenoids affect oxidative processes in biological systems by the quenching of excited states and by the scavenging of radicals.1−6 Carotenoids are lipophilic, and their protective effects are linked to membranes often in synergistic interaction with hydrophilic plant phenols.7,8 As for radical scavenging, radical adduct formation (RAF) competes with electron transfer (ET) and hydrogen atom transfer (HAT).9−13 HAT for β-carotene (β-Car) is only known for the strongly oxidizing hydroxyl radical •OH, while more reducing radicals like the superoxide radical anion O2•− may even be oxidized by some of the least reducing carotenoids like astaxanthin (Ast)4,9 •
•
β‐Car + OH → β‐Car( −H) + H 2O
(1)
Ast + O2•− → Ast•− + O2
(2)
not directly available for scavenging by the phenols or phenolates.14 Carotenoids were recently shown to be optimally regenerated by moderately reducing plant phenolates in agreement with the Marcus theory for electron transfer.8 Nitric oxide (NO•) is involved in numerous biological functions including aqueous phase antioxidant activities.15−20 NO• is a relatively long-lived radical and may be reducing or oxidizing depending on the actual conditions15,16,19
(3)
NO• + H+ + e− → HNO
(5)
Received: July 28, 2014 Revised: September 14, 2014 Published: September 16, 2014
Such regeneration of β-Car results in antioxidant synergism since regenerated β-Car may continue to scavenge lipid radicals © 2014 American Chemical Society
(4)
NO• is in the aqueous phase of mammalian muscles bound to heme pigments like myoglobin,18 which serves as an NO• reservoir. However, NO• is also soluble in lipids and may easily diffuse from one phase to another phase with different polarity. The effect of NO• interaction with lipophilic antioxidants like carotenoids on the overall antioxidant efficiency remains unknown.21 NO• is, however, known to add to β-Car forming nitroxides.22,23 Nitroxides are stable radicals and as such potential antioxidants.
The interaction of β-Car and other carotenoids with hydrophilic antioxidant occurs at lipid/water interfaces, where less efficient water-soluble antioxidants (φ-OH) mainly in the deprotonated form may regenerate β-Car from the β-Car radical cation (β-Car•+) as the oxidation product from scavenging of lipid radicals in the lipid phase2,7 β‐Car •+ + φ‐O− → β‐Car + φ‐O•
NO• + H 2O → NO2− + 2H+ + e−
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calculated using time-dependent DFT (TDDFT) method. The effect of solvent, n-hexane, was included by using the integralequation-formalism polarizable continuum model (IEFPCM)27 in the single-point calculations and TDDFT calculations. After testing six functionals (BPW91, B3PW91, M06-2X, PBE0, CAMB3LYP, and wB97XD), wB97XD functional was chosen, since it predicts the absorption at 439 nm for β-Car and 1015 nm for β-Car radical cation, in good agreement with experimental results.9,28 The geometry optimizations were preformed in the gas-phase with the wB97XD functional and 631G* basis set (labeled as B1). Each stationary point was classified as a minimum or transition state by the frequency calculations. Then the energy was corrected by using a larger basis set, 6-311+G(2d,2p), labeled as B2. The free energies could then be calculated from the B2 single-point electronic energy with addition of Gibbs free energy correction from the frequency calculation and the energy difference between gas and solution phase at the B1 level.
For the complicated pattern of antioxidant interactions at water/lipid interfaces as in membranes, knowledge of NO• reaction with carotenoids is missing. Such interaction may be important for function and integrity of membranes. We have accordingly studied the initial step in the reaction of NO• with β-Car combining experimental methods with theoretical calculations in order to establish stoichiometry of such reactions and to identify effects on both NO• as a bioregulator and on functions of β-Car as a membrane stabilizing antioxidant.24
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MATERIALS AND METHODS Chemicals. All-trans-β-carotene (β-Car) was purchased from Sigma-Aldrich Chemical Co. (St. Louis, MO). NO• (99.95%) was purchased from Yanglilai Chemical Gas Ltd. (Beijing, China). Sodium hydroxide and sodium nitroprusside (SNP, ≥96.0%) were purchased from Beijing Chemical Works (Beijing, China). Anhydrous calcium chloride (≥96.0%) was purchased from Aoboxing Biotechnology Co., Ltd. Beijing, China. HPLC-grade tetrahydrofuran (THF, Mreda Technology Inc., USA), n-hexane (Sinopharm Chemical Reagent Co. Ltd., Shanghai, China), and methanol (Siyou Fine Chemical Co. Ltd. Tianjin, China) were used as received. Reaction of β-Carotene with Gaseous NO•. NO• gas was purified by bubbling through a NaOH (5 M) aqueous solution and dried by anhydrous CaCl2 to remove NO2 and water, respectively, before bubbling into a 2.0 × 10−6 M β-Car n-hexane solution under anaerobic conditions. Steady state UV−vis absorption spectra during the reaction of β-Car with gaseous NO• were recorded in a 1 cm optical cell on a Cary50 spectrophotometer (Agilent Technologies Inc., Santa Clara, CA). Mass spectrometry (LC/MS) of the final product in nhexane was determined using Bruker Apex IV FTMS (Irvine, CA). Reaction of β-Carotene with NO• Formed Photochemically from Sodium Nitroprusside. Binary solvent methanol:THF = 4:1 (v/v) was used to solubilize both lipophilic β-Car (4.0 × 10−5 M) and hydrophilic SNP (1.2 × 10−2 M). Samples in a 1 cm optical cell were exposed to light from a broad spectrum Xe lamp under anaerobic condition for increasing exposure time. β-Car (4.0 × 10−5 M) and SNP (1.2 × 10−2 M) each in methanol:THF = 4:1 (v/v) solution were photolyzed separately and when combined in solution. Laser Flash Photolysis. The laser flash photolysis apparatus has been described elsewhere.9 Briefly, the excitation laser pulses at 355 nm (3−5 mJ/pulse, 7 ns, 10 Hz) were supplied by a Nd3+:YAG laser (Quanta-Ray PRO-230; Spectra Physics, Santa Clara, CA), and the probe light provided by a xenon-flash lamp synchronized to the electric gate of the ICCD detector (L7685, Hamamatsu Photonics, Hamamatsu City, Japan). Solutions of samples were 1.0 × 10−4 M β-Car and 1.2 × 10−2 M SNP and photolyzed immediately after preparation. The optical path length of the flow cuvette (∼20 mL) used for laser flash photolysis was 5 mm. The anaerobic condition was achieved by bubbling the solution with high-purity argon for ∼30 min. All of the measurements were carried out at room temperature (25 °C). Computational Details. The reaction of β-Car and NO• radical was investigated using density functional theory (DFT) with the Gaussian 09 program package.25 All-trans conformation of β-Car was used since it was found to be the most stable isomer by a theoretical study.26 The absorption energies and intensities of the products in all the studied channels were
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RESULTS Nitric oxide (NO•) is known to bleach β-Car, and an excess of NO • corresponding to a NO • /β-Car molar ratio of approximately 10 was for tetrachloromethane as an electron withdrawing solvent found to result in complete bleaching of the visible absorption.22 The bleaching of β-Car indicates that the polyene conjugation in β-Car is disrupted. For n-hexane as solvent, NO• was likewise found to bleach β-Car when added in excess, see Figure 1.
Figure 1. Spectral changes during reaction of β-Car (4.0 × 10−6 M) with NO• in n-hexane at 25 °C for continuing bubbling of gaseous NO• into β-Car solution.The initial curve: the absorption spectrum of β-Car in n-hexane.
The stoichiometry of the reaction of NO• with β-Car became clear from a mass spectrometric analysis of the reaction mixtures, see Figure 2. One major reaction product was found to have a mass of 596.6 corresponding to β-Car with two NO• added (Scheme 1). This compound together with a minor reaction product with the higher mass of 624.6 and many degradation products of lower mass helped to track the main reaction path for the apparent rather complex degradation of βCar exposed to excess of NO•. The reaction of β-Car with NO• was further studied under conditions where NO• was generated in situ photochemically from SNP dissolved in 4:1 methanol:THF under anaerobic conditions,29 using continuous wave photolysis with light from a broad spectrum Xe lamp, and using laser flash photolysis 11660
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Figure 2. ESI mass spectra of the product of β-Car reaction with excess of NO• in n-hexane.
Scheme 1. Mechanism of Initial Reaction of β-Car in nHexane Involving Two NO•
Figure 3. UV−visible absorption spectra of (a) SNP (1.2 × 10−2 M), (b) β-Car (4.0 × 10−5 M), and (c) SNP (1.2 × 10−2 M) in the presence of β-Car (4.0 × 10−5 M) in methanol:THF = 4:1 (v/v) binary solvent as determined using 1 cm optical cell. Samples exposed to broad spectrum Xe lamp under anaerobic condition for increasing exposure time.
Fe(CN)5NO3− and β-Car dissolved, each at the same concentration as for the separate photolysis experiment, clearly showed an increasing bleaching of β-Car (Figure 3c). β-Car accordingly are bleached not only by direct light exposure, a well-known process,31 but also through reaction with a photoproduct of Fe(CN)5NO3− identified as NO• on the basis of spectral characteristics of the other photoproduct, Fe(CN)5(solvent)3−, cf. eq 6. β-Car is almost depleted completely within 6 min. The increasing absorbance at 400 nm for SNP (Figure 4a), corresponding to disappearance of Fe(CN)5NO3− as the result of light irradiation, shows similar dependence on exposure time in the absence of β-Car as in the presence of β-Car except for minor absorption increase at 4−6 min in the presence of β-Car. β-Car does accordingly not interfere with the photochemical formation of NO• from Fe(CN)5NO3−. The product of β-Car reacting with NO• formed from photolysis of Fe(CN)5NO3− decays completely within 10 min. The absorption spectrum of the product formed from reaction of β-Car with NO• may thus be obtained as the difference in absorption spectra between SNP and SNP/β-Car solutions at 6 min irradiation. The product spectrum shows absorption between 350−500 nm as seen in Figure 6 (blue line). In contrast, the rate of absorbance decrease at 480 nm, corresponding to the reaction of β-Car following light exposure, clearly depends on the presence of Fe(CN)5NO3− (Figure 4b). The presence of Fe(CN)5NO3− in the photolysis solution accordingly strongly accelerates the reaction of β-Car adding
(monochromatic at 355 nm in 7 ns pulses) combined with transient absorption spectroscopy for a dynamic characterization. Both methods used the photochemical reaction of eq 6 for a controlled NO• release to facilitate identification of the initial reaction products formed prior to the subsequent degradation reactions: Fe(CN)5 (NO)3 − + solvent → [Fe(CN)5 (solvent)]3 − + NO•
(6)
The steady-state absorption spectra of a solution of SNP (1.2 × 10−2 M) are shown in Figure 3a for increasing Xe lamp irradiation time. The primary photolysis reaction of Fe(CN)5NO3− corresponds to the reaction of eq 6, and the increasing absorption at λmax = 402 nm is similar to what has been observed for aqueous solutions.29,30 β-Car is also sensitive to light from the Xe lamp, and the absorbance intensity of βCar solution (4.0 × 10−5 M) is shown to decrease for increasing irradiation time (Figure 3b). Photolysis of solutions with both 11661
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Figure 6. Red line: difference absorption spectra between SNP/β-Car and SNP in methanol:THF = 4:1 (v/v) binary solvent at 30−50 ns in Figure 5. Black line: absorption spectra at 60 s from Figure 1 of β-Car (4.0 × 10−6 M) bubbled with NO•. Blue line: difference absorption spectra between SNP (1.2 × 10−2 M) and SNP (1.2 × 10−2 M)/β-Car (4.0 × 10−5 M) in methanol:THF = 4:1 (v/v) binary solvent at 6 min exposed to broad spectrum Xe lamp in Figure 3a,c. Green line: absorption spectra of trans-C7,8-β-Car···(NO)2 obtained from DFT theoretical calculation.
Figure 4. Time evolution profilesas obtained from Figure 3 for (a) SNP and SNP in the presence of β-Car at 400 nm, and (b) β-Car and β-Car in the presence of SNP at 480 nm.
further support to the role of the photochemically formed NO• as reagent for β-Car in a reaction parallel to the direct photolysis of β-Car. Laser flash photolysis with nanosecond pulses at 355 nm mainly results in excitation of Fe(CN)5NO3− as may be concluded from the relative absorbance of β-Car and Fe(CN)5NO3− (Figure 3a,b). A transient absorption at 370− 500 nm has a lifetime in the nanosecond time regime (Figure 5a,b). The difference spectrum with maximum at 420 nm (red
Three types of reactions, electron transfer (ET), hydrogen atom transfer (HAT), and radical adduct formation (RAF), which all previously were considered for the reaction of β-Car with the hydroxyl radical •OH, will also be considered for the reaction between NO• and β-Car, as they all will affect the conjugated polyene backbones of β-Car: ET:
β‐Car + NO• → β‐Car•+ + NO−
(7)
HAT:
β‐Car + NO• → β‐Car(− H)• + HNO
(8)
RAF:
β‐Car + NO• → (β‐Car···NO)•
(9)
For the ET reaction, experimental standard reduction potentials for β-Car•+/Car (E° = 1.06 V)11 and NO•, H+/ HNO (E = −0.55 V)15 combined with pKa = 7.2 for HNO,16 indicated that the ET reaction of eq 7 is not possible with ΔG° = 196 kJ mol−1 (Table 1), while the reduction of β-Car•+ by NO− is spontaneous. The ET reaction will accordingly not be considered further in this study. To gain more insight into the reaction mechanism, DFT calculations were performed for the HAT and RAF reactions. We consider only the reactivity of one-half of β-Car, up to C15 (Scheme 1) due to its symmetry. For the HAT reactions, the hydrogen atoms connected to C1a, C2, C3, C4, C5a, C9a, and C13a were considered. For the RAF reactions, the following reaction sites at β-Car were considered: C5, C6, C7, C8, C9, C10, C11, C12, C13, C14, and C15. The calculated absolute energy (in a.u.) for the products formed by the reaction of βCar with one NO• at the B1 level is listed in Table 2, showing that the products for the HAT reactions have much higher energies than that for the RAF reactions. This is in agreement with the larger reaction free energy (ΔG° = 238 kJ mol−1) estimated from BDE for hydrogen abstraction from both β-Car and HNO (Table 1). Therefore, further calculations were accordingly only performed for the RAF reactions. For the RAF reactions, we have optimized the geometries of the reactant complexes, transition states, and product complexes for all of the possible addition paths. The calculated activation free energy and the reaction free energy for addition one NO• to β-Car are given in Table 3. As shown in Table 3, the addition reactions to C5 has the lowest reaction barrier ΔG≠, while the additions to C7 and C9 have reaction barriers close to that of C5 (within 0.5 kJ•mol−1). However, the
Figure 5. Transient absorption spectra at (a) 30−50 ns and (b) 100− 400 ns for SNP (1.2 × 10−2 M) in the presence and absence of β-Car (1.0 × 10−4 M) in methanol:THF = 4:1 (v/v) binary solvent following 355 nm photoexcitation with 7 ns pulse.
line, Figure 6) between the transient spectra of Fe(CN)5NO3− in the absence of β-Car and in the presence of β-Car is tentatively assigned to the absorbance of a transient species formed from β-Car reacting with NO•. The lifetime of this transient species with an absorption maximum at 420 nm is around 100 ns as it decays exponentially with a rate constant k ≈ 107 s−1 at 25 °C. The spectral characteristics of the two species, photochemical intermediate product of β-Car and nitroprusside (blue line in Figure 6) and intermediate product at 60 s (Figure 1) of excess NO• with β-Car (black line in Figure 6) are very similar. 11662
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Table 1. Reaction Free Energies (ΔG°, kJ mol−1) for ET, HAT, and RAF Reaction of β-Car with NO• and •OH ΔG° (kJ mol−1) ET HAT RAF
•
•
reaction mechanism
R = NO
R• = •OH
β-Car + R• → β-Car•+ + R− β-Car + R• → β-Car(−H)• + HR β-Car + R• → (β-Car···R)• (β-Car···R)• + R• → (β-Car···R2)
196a 238c 22(C7)e −11(trans-C7, 8)g
−69b −214d −142(C5)f
a Calculated from the standard reduction potentials for β-Car•+ and NO•, and pKa of HNO from refs 11, 15, and 16. bFrom ref 12. cCalculated from bond dissociation energy (BDE) as reported in refs 10 and 15. dFrom ref 9. eNO• addition to C7 of β-Car; fFrom ref 13. •OH addition to C5. gA second NO• addition to C7.
Table 4. Calculated Acitvation Free Energy (ΔG≠ in kJ mol−1) and Reaction Free Energy (ΔG° in kJ mol−1) of Second NO• RAF Paths after the First NO• Addition on the C7 Site, the Maximum Wavelength (λmax in nm) and Oscillator Strength (f) of the Absorption Spectra of the Corresponding Products
Table 2. Calculated Absolute Energy (in a.u.) for the Products Formed from the Reaction of β-Car with One NO• Molecule at the B1 Level RAF
HAT
site
E(B1)
site
E(B1)
C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15
−1687.229026 −1687.188445 −1687.232015 −1687.204954 −1687.223974 −1687.210503 −1687.221507 −1687.214865 −1687.220180 −1687.218331 −1687.220251
C1a C2 C3 C4 C5a C9a C13a
−1687.142918 −1687.147445 −1687.152866 −1687.194530 −1687.186858 −1687.184895 −1687.182531
Table 3. Calculated Activation Free Energy (ΔG≠ in kJ mol−1), Reaction Free Energy (ΔG° in kJ mol−1) for RAF Paths of First NO• Addition to β-Car, and the Maximum Wavelength (λmax in nm) and Oscillator Strength (f) of the Absorption Spectra of the Corresponding Products
a
site
ΔG≠a
ΔG°a
λmax
F
C5 C6 C7 C8 C9 C10 C11 C12 C13 C14 C15
74.36 160.30 74.40 91.08 74.74 99.57 81.05 92.59 77.92 79.17 76.37
44.48 151.69 21.95 95.01 52.58 86.53 56.76 72.86 63.29 53.30 63.66
494 435 471 423 458 407 434 381 423 360 394
4.07 4.15 3.83 3.76 3.42 2.48 3.09 1.99 2.40 1.82 0.96
sitea
ΔG≠
ΔG°
λmax
f
C8 trans-C8 C10 trans-C10 C12 trans-C12 C14 trans-C14 C7′ C9′ trans-C9′ C11′ trans-C11′ C13′ trans-13′ C15′ trans-C15′
56.46 55.51 57.93 50.55 58.46 51.60 49.99 55.29 54.02 48.18 41.94 53.26 54.69 57.32 59.71 55.95 48.35
−3.77 −10.60 14.23 3.61 9.72 10.65 2.18 7.50 7.00 8.23 8.01 10.43 15.06 16.84 12.36 21.08 6.48
423 425 406 406 385 386 361 359 403 385 392 372 372 361 372 342 347
3.64 3.78 3.34 3.31 2.99 2.98 2.80 2.78 3.81 3.45 3.42 3.15 3.14 2.67 3.14 3.06 2.83
a The prefix “trans-” was used when the second NO• addition occurred in the different direction of the β-Car plane compared with the first NO• addition.
for the trans-addition, suggesting that both additions can be expected to occur. From an inspection of Table 5, it may be concluded that the second NO• addition after the initial addition to the C5 site is endothermic, which reveals that adduct formed by first NO• addition to C5 site is unlikely to be observed by experiments. The formation of dinitrosyl-β-Car accordingly follows the pattern outlined in Scheme 1 leading to the trans-7,8-dinitrosyl-β-Car as the isomer favored. The mononitrosyl-β-Car will be an intermediate of fleeting existence not building up to any significant concentration in homogeneous solution. The energy profile for NO• addition to β-Car is displayed in Scheme 2. This scheme also includes the initial step forming the reaction complexes for the addition of the first and the second NO• prior to the activation step. The reaction begins with the formation of the reactant complex [βCar](NO•), which is calculated to be 26.56 kJ•mol−1 less stable than free β-Car and NO•. The first NO• addition to C7 was favored with a reaction free energy ΔG° = 21.95 kJ•mol−1, an activation free energy ΔG≠=74.40 kJ•mol−1, and a rate constant k=0.56 s−1 calculated using conventional transition state theory (TST).4 The second NO• addition reaction starts with formation of the [β-Car-7-NO•](NO•) adduct and occurs
Including solvent effect (n-hexane).
reaction free energy clearly indicates that C7 should be the preferred reaction site from a thermodynamic point of view. Due to the radical character of mononitrosyl-β-Car, it can further react with the NO radical. Accordingly, we also investigated the second NO• addition reactions after the initial addition at the C5 and C7 sites. From the results summarized in Tables 4 and 5, it can be seen that the reactions for the second NO• additions have lower reaction free energies and barriers compared with that for the first NO• additions. As shown in Table 4, most of the second addition processes after the initial addition to C7 site were found to be endothermic. The exceptions are the cis- and trans-addition to the C8 site. The barrier for the cis-addition is only 1 kJ mol−1 higher than 11663
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initially seen both in the reaction between NO• and β-Car and in the photochemical reaction between Fe(CN)5NO3− and βCar. The reaction of NO• with β-Car and other carotenoids may have significant impact in vivo. The first NO• addition leads to delocalization of the single electron from NO• along the conjugated polyene backbone, which may relate to antioxidant activity of β-carotene toward NO•. The dinitrosyl adduct of βCar seems to have some stability as a nonradical and may carry NO• in lipophilic parts of membranes. The further reaction of β-Car with NO• will rather lead to nonreversible fragmentation for larger excess of NO•. The fragmentation pattern of β-Car seen for large excess of NO• in n-hexane indicates increments in molecular weight between fragments of 28 m/z, which could be explained by formation of stable nitroxide radicals from a fragment formed by cleavage between C7 and C8 in the initially formed C7/C8 dinitrosyl adduct (Scheme 3). As seen from the mass spectrum of the reaction mixture of βCar and NO•, the fragment peaks with m/z values ranging from 456.4 to 624.8 are evenly separated with increments of 28 m/z between each two adjacent peaks. This group of fragment peaks can be assigned as follows. The conjugated chain of β-Car undergoes cleavage of the double bond C7C8, and the larger fragment has a molecular weight of 400 m/z, which is in agreement with the fragment peak seen in the mass spectrum with a m/z value at 400.4. The addition of NO• to the β-Car fragment may be initiated by hydrogen atom abstraction as shown in Scheme 3. The net increase in molecular weight of the fragments seen in Scheme 3 is 28 m/z, which is in agreement with the interval of the m/z values of the fragment peaks from 456.4 to 624.8 of the mass spectrum. The stable nitroxide radical may further react with more NO• in the presence of excess NO•. This should give an explanation for the mass spectrum of the radical addition products of β-Car and NO•, while the variance in relative abundances of each fragment peak may be related to the stoichiometry of the reactions between β-Car and NO•. The initial mononitrosyl adduct of the fragment with a molecular weight of 428 m/z is not seen in the mass spectrum, but the fragment peaks with m/z values ranging from 456.4 to 624.8 may be assigned to the adduct products of 2 to 8 NO• to the 400 m/z fragment (Scheme 3).
Table 5. Calculated Activation Free Energy (ΔG≠ in kJ mol−1) and Reaction Free Energy (ΔG° in kJ mol−1) of Second NO• RAF Paths after the First NO• Addition on the C5 Site, the Maximum Wavelength (λmax in nm) and Oscillator Strength (f) of the Absorption Spectra of the Corresponding Products sitea
ΔG≠
ΔG°
λmax
f
C8 trans-C8 C10 trans-C10 C12 trans-C12 C14 trans-C14 C7′ trans-C7′ C9′ trans-C9′ C11′ trans-C11′ C13′ trans-13′ C15′ trans-C15′
53.76 54.95 60.03 52.19 59.97 50.63 50.45 62.52 57.60 50.10 53.89 55.05 48.28 51.44 58.24 49.82 48.47 57.17
3.78 11.43 18.94 11.84 23.64 5.46 9.50 19.13 4.27 11.40 14.87 25.35 14.20 17.85 31.94 21.89 5.92 17.28
425 421 405 406 387 386 363 363 482 418 408 410 394 397 381 385 365 364
3.76 3.77 3.36 3.43 3.11 3.11 3.20 3.15 2.97 3.91 3.53 3.60 3.13 3.19 2.71 2.83 2.74 2.77
The prefix “trans-” was used when the second NO• addition occurred in the different direction of the β-Car plane compared with the first NO• addition. a
through trans-addition at C8 with ΔG° = −10.60 kJ•mol−1 and ΔG≠ = 55.51 kJ•mol−1. β‐Car + 2NO• ⇄ β‐Car(7,8‐(NO)2 )
(10)
•
The concentration of NO in n-hexane is higher than the concentration of β-Car and a significant concentration of βCar(7,8-(NO)2) will appear.32 The further reactions as are evident from the MS will remove the dinitrosyl product and force the reaction of eq 10 to the product side. The calculations further indicate an absorption maximum for the dinitrosyl addition product with λmax = 425 nm (green line, Figure 6). The spectra agree reasonably well with the absorption observed by transient absorption spectroscopy following laser flash photolysis of mixture of β-Car and SNP, and with the absorption observed both during the photochemical reaction of β-Car with SNP and during the reaction of excess NO• with β-Car (Figure 6). The theoretical calculations accordingly provide evidence for the nature of the reaction intermediate
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DISCUSSION Nitric oxide, NO•, is a radical which like the hydroxyl radical, • OH, has high molecular mobility. The two radicals, however, function differently in biological systems due to significant differences in reactivity. •OH has been shown to react with β-
Scheme 2. Reaction Energy Diagram of β-Car with NO• Forming the C7,8 Adducta
a
Energies based on DFT-calculation are given in kJ mol−1. 11664
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Scheme 3. Suggested Mechanism for NO• Addition to β-Car Fragment Formed Following C7/C8 Carbon Bond Cleavage in n-Hexane during Reaction with Excess NO•a
mononitrosyl with a large negative ΔG° will favor C7 addition over C5 addition for NO•. It seems accordingly safe to conclude that the initial NO• addition to β-Car result in enhancement of reactivity of β-Car toward NO• and probably also other radicals. Such phenomena has previously been related to functions of carotenoids as molecular wires in membranes releasing oxidative stress from membrane surface to lipophilic antioxidants in membrane interior.24 The importance of the NO• interaction with β-Car is still speculative, but the facile stepwise addition of NO• to carotenoids deserves further attention. The initial addition of two NO• to β-Car is followed by degradation of β-Car through further reaction with NO• as suggested in Scheme 3. The initial two NO• addition reactions seem according to the theoretical calculation to be driven by the further degradation. The stricking consistency in a difference of 28 m/z between various reaction products resulting from the reaction between β-Car and excess NO• finds an explanation through addition of an increasing number of NO•. In contrast to the two initially added NO•, further addition seem to involve abstraction of hydrogen atoms, in agreement with the increasing mass of 28 m/z corresponding to addition of NO• and abstraction of two hydrogen atoms (14 + 16 − 2) (Scheme 3). The mechanism behind abstraction of hydrogen is not obvious, but NO• may disproportionate in the reaction mixture under the influence of catalytically active reaction intermediates:33 4NO• → N2 + 2NO2
(11)
NO2, as resulting from such disproportionation reactions, is known to abstract hydrogen atoms from β-Car.34,35 The nitroxide derivatives formed through reaction of NO• with βCar may serve as antioxidants in membranes being formed through diffusion of NO• into the lipophilic membrane for reaction with β-Car.
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CONCLUSIONS The reaction of NO• with β-Car seems to depend on the level of NO•. For reaction of β-Car with NO• for low levels of NO•, the reactivity of mononitrosyl adducts toward radicals is higher than for β-Car itself. For higher concentrations of NO•, β-Car may form lipophilic nitroxide derivatives with good antioxidant capabilities through further addition of NO•.
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a
AUTHOR INFORMATION
Corresponding Authors
*E-mail: [email protected]. Tel: +86-10-6251-6604. Fax: +86-10-6251-6444. *E-mail: [email protected]. Tel: +45-3528-3221. Fax: +45-35283344.
Stereochemistry of intermediates and products are unknown.
Car through the HAT mechanism abstracting a hydrogen atom from β-Car with a large negative free energy of reaction resulting in formation of the neutral β-Car radical, β-Car(-H) • (Table 1).9 Other radicals including NO• will not react with βCar through a HAT reaction. For NO• and β-Car, ΔG° for HAT, as calculated from BDE for β-Car and HNO, is strongly positive, a result confirmed by the present DFT calculations. According to available thermodynamic data, NO• will not oxidize β-Car to β-Car•+.15 In contrast, NO• will react with βCar through RAF as is already known from experiments22,23 and now confirmed by DFT calculations. The ΔG° = 21.95 kJ mol−1 of Table 1 refers to addition of NO• to C7, while ΔG° = −142 kJ mol−1 refers to addition of •OH to C5 of β-Car. However, the subsequent addition of NO• to C8 in the
Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS This work has been supported by the Fundamental Research Funds for the Central Universities, and the Research Fund of Renmin University of China (RUC No. 10XNI007, 14XNLQ04, 12XNLJ04, 14XNH058). LHS thanks the Danish Research Council for Independent Research, Technology and Production Science continuing support currently as the Grant 09-065906/FTP: Redox Communication in the digestive tract. 11665
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