Light-Induced Oxidation of Unsaturated Lipids as Sensitized by

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J. Phys. Chem. B 2010, 114, 5583–5593

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Light-Induced Oxidation of Unsaturated Lipids as Sensitized by Flavins Kevin Huvaere,† Daniel R. Cardoso,‡ Paula Homem-de-Mello,§ Signe Westermann,† and Leif H. Skibsted*,† Department of Food Science, Faculty of Life Sciences, UniVersity of Copenhagen, RolighedsVej 30, DK-1958, Frederiksberg C, Denmark, Departamento de Quı´mica e Fı´sica Molecular, Instituto de Quı´mica de Sa˜o Carlos, UniVersidade de Sa˜o Paulo, AVenido Trabalhador Sa˜o Carlense 400, CP 780, CEP 13560-970, Sa˜o Carlos-SP, Brazil, and UniVersidade Federal do ABC, Rua Santa Ade´lia 166, Bairro Bangu, CEP 09210-170, Santo Andre´-SP, Brazil ReceiVed: December 25, 2009; ReVised Manuscript ReceiVed: March 1, 2010

Triplet-excited riboflavin (3RF*) was found by laser flash photolysis to be quenched by polyunsaturated fatty acid methyl esters in tert-butanol/water (7:3, v/v) in a second-order reaction with k ∼ 3.0 × 105 L mol-1 s-1 at 25 °C for methyl linoleate and 3.1 × 106 L mol-1 s-1, with ∆H‡ ) 22.6 kJ mol-1 and ∆S‡ ) -62.3 J K-1 mol-1, for methyl linolenate in acetonitrile/water (8:2, v/v). For methyl oleate, k was 0 for electron transfer. Interaction of methyl esters with 3RF* is considered as initiation of the radical chain, which is subsequently propagated by combination reactions with residual oxygen. In this respect, carbon-centered and alkoxyl radicals were detected using the spin trapping technique in combination with electron paramagnetic resonance spectroscopy. Moreover, quenching of 3RF* yields, directly or indirectly, radical species which are capable of initiating oxidation in unsaturated fatty acid methyl esters. Still, deactivation of triplet-excited flavins by lipid derivatives was slower than by proteins (factor up to 104), which react preferentially by electron transfer. Depending on the reaction environment in biological systems (including food), protein radicals are expected to interfere in the mechanism of light-induced lipid oxidation. Introduction Riboflavin (RF), generally referred to as vitamin B2, and its derivatives are among the most important photosensitizers in biological systems and are capable of inducing photooxidation of a variety of biomolecules.1,2 Flavins operate as a photosensitizer through a direct chemical quenching of the corresponding singlet or triplet-excited states by substrates (type I mechanism) or through a physical quenching of the triplet-excited state by ground state oxygen to yield singlet oxygen, 1O2 (type II mechanism).3,4 Type I photooxidation depends on initial electron or hydrogen atom transfer from suitable substrates, for example, amino acids or flavonoids,2,5,6 to the excited flavin followed by, in the presence of oxygen, superoxide (O2•-) production with concomitant regeneration of riboflavin.7,8 For type II photooxidations, 1O2 is the reactive intermediate with the potential of adding to electron-rich sites, such as double bonds in unsaturated lipids, to yield hydroperoxides or to oxidize riboflavin now becoming the substrate.9,10 Endogenous lipid oxidation is closely related to oxidative stress and is possibly involved in the development of various pathological conditions.11 Moreover, preservation and nutritive * To whom correspondence should be addressed. E-mail: [email protected]. Tel: + 45 35 33 32 21. Fax: + 45 35 33 33 44. † University of Copenhagen. ‡ Universidade de Sa˜o Paulo. § Universidade Federal do ABC.

value of foods rich in polyunsaturated lipids is compromised by oxidative instability, eventually leading to formation of toxic reaction products.12,13 The initiation step of the radical chain accounting for lipid autoxidation is not well characterized but probably depends on enzymatic activity, catalysis by transition metals, or exposure to irradiation including light. As for the latter, sensitizing is essential since lipids absorbs little light in the visible or UVA region. Still, details of light-induced radical initiation remain elusive due to lack of kinetic and thermodynamic data for crucial reaction steps occurring during early events. Thus, a systematic approach with focus on flavin derivatives as photosensitizers was conceived to investigate the reactivity of singlet and triplet-excited states toward model lipid systems (i.e., methyl esters of fatty acids). Theoretically calculated thermodynamic properties were combined with kinetic parameters extracted from time-resolved laser flash spectroscopy. Incipient lipid radicals were characterized by electron paramagnetic resonance (EPR) spectroscopy using the spin-trapping technique for further mechanistic insight. The goal of these combined studies was to elaborate mechanistic details of the free radical pathways accounting for lipid oxidation during light exposure. The importance of such mechanism has largely escaped attention, particularly since sensitized lipid reactions have been commonly associated with nonradical peroxidation by singlet oxygen.

10.1021/jp9121744  2010 American Chemical Society Published on Web 04/08/2010

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Experimental Section Chemicals. Methyl stearate, methyl oleate, methyl linoleate, and oleyl alcohol were purchased from Fluka (Buchs, Switzerland). Methyl linolenate, lumiflavin, 3-methylindole, tertbutanol, ferrocene (Fc), tetrabutylammonium perchlorate, and the spin traps 5,5-dimethylpyrroline N-oxide (DMPO) and 2-methyl-2-nitrosopropane (MNP) were supplied by SigmaAldrich (St. Louis, MO), while 2,2-dimethyl-4-phenyl-2Himidazole 1-oxide (DMPIO) was obtained from Alexis Chemicals (Lausen, Switzerland). Acetonitrile (Lab-Scan, Dublin, Ireland) was of spectrophotometric grade, while aqueous solutions were prepared using purified water (18 MΩ · cm) from a Milli-Q purification system (Millipore, Bedford, MA). Electron Paramagnetic Resonance Spectroscopy. Samples for EPR analyses were prepared by dissolving the appropriate substrate (in a concentration varying from 10 mM up to 200 mM, except for methyl stearate, which was only soluble up to ∼20 mM) and lumiflavin (25 µM) in acetonitrile (or an acetonitrile/water mix for specific experiments). After extensive purging with nitrogen (20 min), the spin trap of choice (DMPO, DMPIO, or MNP) was added to reach a final concentration of 5 mM (DMPIO) or 10 mM (DMPO and MNP). Samples were then loaded into a flat quartz cell and subjected to an additional nitrogen purging step (5 min). Subsequent irradiation was carried out inside the EPR spectrometer cavity with a Photonics Polychrome II unit (TILL Photonics, Gra¨felfing, Germany), which allowed continuous excitation of the reaction mixtures at 440 nm (power ∼2.5 mW). EPR spectroscopy was performed on a Bruker ECS 106 spectrometer (Bruker, Karlsruhe, Germany), applying the following settings: center field, 3475 G [Gauss]; sweep width, 80 G; microwave power, 10 mW; modulation frequency, 100 kHz; modulation amplitude, 1.0 G (or 0.25 G to improve resolution of the intense EPR signals resulting from DMPO spin trapping); conversion time, 40.96 ms; time constant, 20.48 ms. Accumulation of 4 scans resulted in the final spectrum of the spin adducts. Simulation and fitting of EPR spectra to calculate hyperfine coupling constants, were performed by the PEST WinSIM program.14 Laser Flash Photolysis. Laser flash photolysis experiments were carried out with an LKS.50 spectrometer from Applied Photophysics Ltd. (Leatherhead, United Kingdom) using the third harmonic (355 nm) of a pulsed Q-switched Nd:YAG laser with 10 ns resolution (Spectron Laser System, Rugby, United Kingdom) attenuated to 10 mJ cm-2 as the excitation source. A R928 photomultiplier tube from Hamamatsu Photonics (Hamamatsu City, Japan) was used to detect the transient absorption (300-800 nm). Appropriate UV cutoff filters were used to minimize the sample degradation by the monitoring light. Samples were excited in 0.5 cm × 1.0 cm fluorescence cuvettes from Hellma (Mulheim, Germany). Each kinetic trace was averaged 16 times and observed rate constants were determined by fitting the data with MatLab R2008 (The MathWorks, Natick, MA). All measurements were performed on fresh solutions thermostatted at 298 ( 0.5 Κ and purged with N2 for 15 min before the experiment. Steady-State Fluorescence Spectroscopy. Fluorescence measurements were carried out using a Hitachi F-7000 Fluorescence Spectrometer (Hitachi High-Tech, Tokyo, Japan) at 298 K in a thermostatted cell holder. The samples were excited in 1.0 cm × 1.0 cm fluorescence cuvettes from Hellma (Mulheim, Germany). Emission spectra (2.0 nm band-pass on the excitation and 4.0 nm on the emission monochromator) were corrected for instrument response and were recorded for excitation at 445 nm. The static fluorescence quenching of

Huvaere et al. singlet-excited riboflavin was studied only for compounds that show the ability to quench the triplet-excited state. All measurements were performed in triplicate. Electrochemistry. Cyclic voltammetry was carried out in a Voltalab PGZ402 electrochemical station (Radiometer Analytical, Lyon, France) connected to a personal computer using the proprietary software Voltamaster 2. Electrochemical oxidations were carried out in acetonitrile solutions using tetrabutylammonium perchlorate (0.1 M) as supporting electrolyte and a three-electrode Pyrex glass with degassing facilities for purging with high purity nitrogen. The working electrode was a 0.5 cm × 0.5 cm boron-doped diamond film deposited on a silicon wafer produced by the Centre Suisse de Electronique et de Microtechnique SA (Neuchatel, Switzerland). The auxiliary electrode was a 2 cm2 platinum foil, and unless otherwise stated, all measurements were carried out using Fc+/Fc as internal quasi-reference electrode. Computational Methods. The DFT calculation procedures are as follows. First, all structures were optimized by the DFT method with the B3LYP functional15–17 and the DGDZVP basis set, in acetonitrile solution simulated with Integral Equation Formalism as Polarizable Continuum Model (IEF-PCM).18–20 The absence of imaginary frequencies was used as a criterion to ensure that the optimized structures represent the minimum of the potential energy surface. Bond dissociation energy (BDE) was calculated by use of the expressions presented by DiLabio et al.21 The lowest triplet excitation energies (ET1) of riboflavin and unsaturated fatty acids methyl ester were estimated by timedependent DFT (TD-DFT).22 Vertical electron affinities (VEA), hydrogen atom affinity (HAA), and vertical ionization potentials (VIP) of riboflavin and unsaturated fatty acids methyl ester were calculated by using a combined DFT method using B3LYP/631+G(d,p) to perform single-point calculation using B3LYP/ 6-31G(d,p) optimized geometries.23 All calculations were performed with the Gaussian 03 code.24 Results and Discussion Kinetics and Thermodynamics of Quenching. The isoalloxazine moiety is responsible for light sensitivity of flavin derivates (see Figure 1 for structures) and the corresponding high molar absorptivities (>104 M-1 cm-1) are characteristic for the π-π* transitions following irradiation.25 Thus, lower singlet-excited states of flavins are populated and subsequently converted to the lowest triplet state by an efficient intersystem crossing (isc), which competes with deactivation via fluorescence. The difference in multiplicity between the singlet-excited and the triplet-excited state significantly affects the respective lifetimes, but still both states were considered as potential oxidants of unsaturated lipids. As for the singlet-excited state of riboflavin (1RF*), steady-state fluorescence experiments were carried out to investigate interactions with model lipid systems. Since the absorption spectrum of flavins in aqueous solution consists of four peaks centered at 446, 375, 265, and 220 nm, exposure to blue light was particularly suitable for excitation. The resulting fluorescence, characterized by intense emission around 520 nm (Φ ) 0.26, τ ) 5.75 ns), is known to be quenched by electron transfer reactions with various electron donors,26 but no interaction was observed in the presence of the various fatty acid methyl esters. Unlike for the singlet-excited state, quenching of tripletexcited riboflavin (3RF*; Φisc ) 0.67, τ ) 27.3 µs in tertbutanol/water 7:3, v/v) was investigated by time-resolved laser flash photolysis with a 355 nm nanosecond excitation pulse.

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Figure 1. Structures of methyl esters of relevant unsaturated fatty acids (including BDE values from DFT calculations) and flavin derivatives. Contour plots of the highest occupied virtual orbital (HOMO) of the methyl esters and the lowest single-occupied orbital (SOMO) of triplet-excited riboflavin (optimized structures) are depicted in the right panel.

Density functional theory (DFT) calculations confirmed previous findings27 that the triplet-excited state is best described as a biradical with unpaired electron spin density centered at the N(1) and (N5) atoms (SOMO-103 ) -0.19 eV and SOMO-104 ) -0.09 eV). The transient absorption spectrum was characterized by maxima around 300, 380, 520, and 720 nm and a strong photobleaching around the 446 nm ground-state absorption (Figure 2A, inset). Decay of 3RF* in the presence of unsaturated fatty acid esters or the milk protein β-casein, monitored at 720 nm, was accelerated with rates proportional to the substrate concentration (Figure 2). Pseudofirst-order rate constants for quenching, extracted from exponential decay traces, were plotted as function of ester or β-casein concentration and the slope of the resulting linear dependence furnished second-order rate

constants (Table 1). Since addition of various concentrations of methyl oleate (up to 35 mM) to a solution containing 70 µM of riboflavin (in tert-butanol/water 7:3, v/v) did not affect decay of 3RF*, the rate constant k must be 0 for the reaction with 1 RF* as electron acceptor corroborates the observed insensitivity of fluorescence to the presence of the esters. Likewise, the change in standard free energy was unfavorable (∆G°ET > 0) for electron transfer to 3RF* (Table 1), despite the fact that quenching was observed with laser flash photolysis. Thus another mechanism must be operating and, in this respect, second-order rate constants (Table 1) for photooxidation are in agreement with literature values for a hydrogen atom transfer process rather than with an outer-sphere electron transfer process.30 The corresponding free energy change was calculated by applying a modified Rehm-Weller equation (eq 3) which takes into account the difference in acidity between the reduced riboflavin radical (pKa ∼ 8.3)31 and radical cations derived from fatty acid esters, of which pKa values were calculated by density functional theory (DFT) (Table 2). Accordingly, it was concluded that hydrogen atom transfer from unsaturated lipid chains to 3RF* was an exergonic process, that is, ∆G°HAT < 0 (Table 1). The acidity of the lipid cation radical is thus shown to be the thermodynamical driving force of the oxidation, but it is unclear whether this intermediate occurs as such in the reaction mechanism. In this respect, the pronounced negative ∆S‡ for the linolenate reaction suggests major rearrangements of the solvent shell and implies formation of a charged activated complex due to incipient ionization. Still rate constants are too low to account for pure electron transfer, hence a concerted mechanism involving an electron and a proton, formally described as hydrogen transfer, is proposed.

Figure 2. Time traces for decay of triplet-excited riboflavin, monitored at 720 nm for (A) increasing concentrations of methyl linolenate, that is, 0 (*), 3.2 (O), 6.4 (b), and 10.7 ([) mM, in tert-butanol/water (7: 3, v/v) under anaerobic conditions. (Inset) Transient difference absorption spectrum obtained after 2 µs for 10.7 mM of quencher. (B) Increasing concentrations of β-casein, that is, 0 (×), 147 (*), 337 (b), and 550 ([) µM, in aqueous phosphate buffer pH 6.4 (I ) 0.16 M). (Inset) Dependence of observed pseudofirst-order rate constant as function of β-casein concentration. The second-order rate constant is obtained from the slope of the linear plot.

Figure 3. Structures of nitrone (DMPO and DMPIO) and nitroso (MNP) spin traps.

The change in standard free energy for photoinduced electron transfer from unsaturated fatty acid esters to the singlet or tripletexcited state of flavin derivatives, ∆G°ET, was calculated by substituting the appropriate parameters in the Rehm-Weller equation (eq 2)

∆G°ET ) 96.48(Eox - Ered - e2/εa) - ∆E0,0

(2)

in which e2/εa, the so-called Coulombic term, can be neglected in solvents with high dielectric constant. ∆E0,0 is the energy level of the singlet or triplet-excited state of riboflavin (239.3

∆G°HAT ) 96.48(Eox - Ered - e2/εa) - ∆E0,0 2.303RT[pKa(RFH · ) - pKa(LH · +)]

(3)

When time-dependent DFT methods were used for determining thermodynamical parameters, it was shown that the theoretically lowest triplet energy of the unsaturated fatty acid esters is significantly higher than that of the flavin derivatives (Table 2), which refutes the possibility of direct energy transfer. Photoinduced electron transfer, on the other hand, depends on the VEA of the triplet-excited flavin and the VIP of unsaturated fatty acid esters (Table 2), which were also calculated by DFT. Summations of the flavin VEA with the VIP of methyl oleate, methyl linoleate, and methyl linolenate, respectively, are positive, supporting the finding that outer-sphere electron transfer as deactivating pathway is thermodynamically unfavorable (∆G°ET > 0). Hydrogen atom transfer (HAT) was determined by HAA of the lowest triplet-excited state of the flavins and the allylic C-H BDE of the unsaturated fatty acid derivatives (Table 2). Particularly bisallylic C-H bonds, as in methyl linolenate, are weak, which coincides with high spin density and a substantial contribution to the HOMO (Figure 1). Summation of the flavin HAA and the C-H BDE of a selected fatty acid derivative is negative, supporting a thermodynamically favorable hydrogen atom transfer (∆G°HAT < 0). This corroborates the free energy change calculations according to the extended Rehm-Weller equation (vide supra). The corresponding thermodynamic cycle (Scheme 1) shows the relation between ∆G°HAT and ∆G°ET, that is, ∆G°HAT ) ∆G°ET + ∆G°H+, in which ∆G°H+ ) -46.6 kJ mol-1 for the linoleate reaction

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TABLE 1: Second-Order Rate Constants at 298.5 ( 0.5 K Obtained for Quenching of Triplet-Excited Riboflavin by Unsaturated Fatty Acid Methyl Esters and β-Casein, Including Activation Parameters for Methyl Linolenatea substrate

k (L mol-1 s-1)

C18:1 C18:2d C18:3e C18:3d β-caseinf

nqb 3.0 ( 0.9 × 105 3.1 ( 0.4 × 106 1.1 ( 0.3 × 106 4.9 ( 0.2 × 108

∆H‡ (kJ mol-1)

22.6 ( 0.8

∆S‡ (J K-1 mol-1)

Eox (V vs NHE)

∆G°ET(kJ mol-1)

∆G°HAT (kJ mol-1)

-62.3 ( 1.2

2.12c 2.06c 2.05c 2.05c

+24.3 +18.5 +17.6 +17.6

-22.7 -28.5 -29.4 -29.4

a

Estimated one-electron oxidation potential (Eox) of fatty acid derivatives are used for calculation of free energy changes for electron and hydrogen atom transfer according to the Rehm-Weller equation. b nq ) no quenching. c Calculated from the empirical formula for alkenes (Eox ) (0.827 × IP - 5.40) + 0.58) reported by Lijser et al.32 and by using the experimental vertical ionization energy.28 d tert-butanol/water (7:3, v/v). e Acetonitrile/water (8:2, v/v). f Aqueous phosphate buffer pH 6.4 (0.16 mol L-1).

TABLE 2: Theoretical Values (Obtained by DFT)a Including VEA, ET1, and HAA As Determined for Triplet-Excited Riboflavin and ET1, VIP, and BDE As Determined for the Weakest C-H Bond in Unsaturated Fatty Acid Methyl Esters, and pKa Values of Their Respective Cation Radicalsb Substrate ET1 (kJ mol-1) VEA (kJ mol-1) VIP (kJ mol-1) BDE (kJ mol-1) HAA (kJ mol-1) C18:1 C18:2 C18:3 flavin

439.5 329.8 266.8 176.6

-518.4

637.2 623.0 617.6

∆G°ET (kJ mol-1) ∆G°HAT (kJ mol-1)

pKa -0.12 -0.12c -0.12c 8.3d c

330.7 293.8 293.8 444.96

118.8 104.6 99.2

-114.3 -151.2 -151.2

a Acetonitrile solution simulated with integral equation formalism as polarizable continuum model. b Corresponding free energy changes for electron and hydrogen atom transfer were calculated according to presented values. c Theoretically estimated pKa for the radical cation LH+•. d pKa value for the radical RFH•.31

SCHEME 1: Thermodynamic Cycle for the Electron Transfer and the Formal H-Atom Transfer Process According to DFT Calculationsa

a The pathway with initial proton transfer was not considered due to low acidity of LH and lack of basic properties of 3RF*.

according to DFT. As the BDE for the allylic C-H in methyl oleate is considerably higher than for bisallylic C-H bonds in methyl linoleate and methyl linolenate (Figure 1), the rate for hydrogen atom abstraction probably becomes too low to be efficient. On the other hand, despite the identical values for the lowest BDE in the two polyunsaturated methyl esters, very different rate constants were obtained due to doubling of reactive bisallylic hydrogens in methyl linolenate. Detection of Radical Intermediates. Although hydrogen abstraction from the (bis)allylic positions of the fatty acid derivatives suggests formation of lipidlike radicals (LH f L•), these could not be detected during flash photolysis experiments with transient absorption spectroscopy. Most likely, the absorbance of radicals from unsaturated fatty acid esters at 280-320 nm was not observed due to intensity of the flavin absorption (data not shown). Still, in an attempt to gain structural information of such species, spin traps (Figure 3) were added to the reaction mixture followed by detection by EPR. Because of poor aqueous solubility of fatty acid derivatives and some of the spin traps, irradiation experiments were carried out in acetonitrile. Spin Trapping by 5,5-Dimethyl-1-pyrroline N-oxide (DMPO). DMPO was selected for initial experiments, as its properties of trapping of (among others) oxygen-centered and carbon-centered radicals have been extensively studied. Thus, after exhaustive purging with nitrogen (20 min), a mixture containing lumiflavin (LF, a flavin derivative soluble in acetonitrile) and DMPO was irradiated continuously at 440 nm, but,

Figure 4. Experimental (upper) and simulated (lower) traces of EPR signals (except for panel B) of DMPO adducts detected during 440 nm irradiation of a nitrogen-purged lumiflavin solution, in the absence and presence of various fatty acid methyl esters. (A) No substrate present; (C) 200 mM methyl oleate; (D) 200 mM methyl linoleate (to improve spectral resolution, EPR analyses of adducts derived from methyl oleate and methyl linoleate were recorded with a modulation amplitude of 0.25 G). Signals presented in panel B refer to the following experiments: (a) irradiation of a mixture of lumiflavin and DMPO (in acetonitrile) under atmospheric (i.e., oxygen-rich) conditions; (b) irradiation of a nitrogen-purged mixture of lumiflavin and DMPO in acetonitrile/water (1:9, v/v); (c) a relative comparison between intensities of the superoxide adduct generation after irradiation (1 min) in the absence (red line) and the presence (black line) of a suitable electron donor (i.e., 3-methylindole). Vertical arbitrary scale indicates intensities of the respective signals.

already in the absence of substrate, an intense spectrum was observed after short exposure (20 min) of Various Fatty Acid Methyl Esters (∼200 mM) in the Presence of Lumiflavin (25 µM) and DMPO (10 mM) compound

RAa

aNb

aHc

aHγ

no substrate methyl stearatef methyl oleate

100 100 76 22 2 62 20 17 1

12.9 12.9 12.9 13.3 13.4 12.9 13.1 13.5 13.6

10.5 10.3 10.4 8.0 ndg 10.5 7.9 7.9 n.d.

1.3 1.3 1.3 1.6 n.d. 1.3 1.6 1.5 n.d.

methyl linoleate

d

SAe i i i i xiiih

a Relative abundance (%). b Coupling constant to DMPO nitrogen (Gauss). c Coupling constant to the DMPO β-hydrogen (Gauss). d Coupling constant to the DMPO γ-hydrogen (Gauss). e Spin adduct identification (Schemes 2 and 3). f Because of low solubility of methyl stearate, concentration in the photoreaction mixture only amounted 20 mM. g nd: not detected. h Spin adducts with regioisomers of the alkoxyl radical ix (Scheme 3) are expected to show similar coupling constants and are virtually indistinguishable.

a Observed spin adducts (with varying spin traps) are depicted in boxes.

lumiflavin (3LF*), readily reduces residual oxygen (Scheme 2).35 The resulting superoxide is stabilized in acetonitrile,36 hence addition to DMPO (i; via the more reactive •OOH) is favored over disproportionation reactions. The latter are more important in aqueous solutions (k ∼ 7.6 × 105 L mol-1 s-1 for •OOH + • OOH, and k ∼ 8.9 × 107 L mol-1 s-1 for O2•- + •OOH),37 which nicely corroborates the lack of detectable EPR signal upon addition of water to the irradiation mixture (Figure 4B). The feasibility of i formation by pathways different from radical addition was also considered, mainly because an efficient electron donor for formation of the pivotal LF•- is lacking in the absence of substrate. Focus was on the interference of 1O2, which was previously shown to interact with DMPO.38 But, setting experimental conditions in favor of 1O2 formation (i.e., irradiation under oxygen-rich atmosphere) only resulted in a weak DMPO signal (Figure 4B). This corroborates the findings by Bilski et al. that formation of i by 1O2 only exceptionally occurs in acidic medium.39 Direct oxidation of DMPO by tripletexcited flavins was refuted as well,40 therefore generation of i was attributed unequivocally to O2•- or •OOH trapping. Formation of LF•-, essential for oxygen reduction, in the absence of “suitable” electron donors was suggested to result from lumiflavin autoreactions, such as triplet-triplet annihilation or triplet-ground state interaction, during continuous irradiation.41 The concurrently formed LF•+, which is a stronger electron acceptor than its triplet-excited predecessor (E ∼ 2.3 vs 1.7 V, respectively),42–44 is less acidic than •OOH in aqueous systems (pKa LF•+ ∼ 6.0 vs pKa •OOH ∼ 4.7).45,46 However, in aprotic solvents a different equilibrium is expected with a shift to noncharged intermediates as result. The observed DMPO adduct was therefore preferably assigned to trapping of the more reactive hydroperoxyl radical (i, Scheme 2) instead of the unreactive O2•-. Addition of 3-methylindole, an electron donor representing the tryptophan side chain,47,48 to the lumiflavin solution was more effective in producing LF•- and, consequently, superoxide concentrations (or hydroperoxyl after protonation) were raised

considerably (Figure 4B). According to our kinetic analyses, methyl linoleate quenches triplet-excited flavins at pronounced lower rates compared to 3-methylindole (3.0 × 105 L mol-1 s-1 vs 2.8 × 109 L mol-1 s-1).47 Still, substituting the fatty acid derivative for 3-methylindole in above experiments (to reach a mixture of 20 mM methyl linoleate, 25 µM lumiflavin, and 10 mM DMPO), resulted in a strong EPR signal from i that eventually hampered detection of other species. However, increasing methyl linoleate concentrations (up to 200 mM) led to the appearance of 2 new spin adducts (Table 3). Their parameters were typically associated with trapping of a fatty acid-derived alkoxyl radical, LO•,49–51 which most likely resulted from decay of the preceding peroxyl radical, LOO• (Scheme 3). The latter results from combination of an incipient carbon radical with residual oxygen at nearly diffusion-controlled rates,52 but, despite previous claims,49,53 addition of lipid-derived peroxyl radicals to DMPO is undetectable.54,55 According to kinetic analyses, methyl oleate (which lacks a bisallylic position) fails to reduce triplet-excited flavins. Nevertheless, irradiation produced an intense, nearly identical EPR spectrum as observed for methyl linoleate photooxidation, including signals from i and an adduct with an alkoxyl radical (aN ∼ 13.3 G, aH ∼ 8.0 G, and aHγ ∼ 1.6 G). The origin of LO• was unclear and the possibility of 1O2 interference in radical formation was investigated by irradiating under atmospheric conditions (20% oxygen), but, apart from line broadening, spectra remained unchanged. Also superoxide, the main reactive oxygen species detected, was considered, but, contrary to what its name leads to suspect, it is a poor oxidant unable to abstract hydrogen from a (bis)allylic position.56,57 Its conjugated acid, • OOH, is a stronger oxidant (E° ∼ 1.48 V),58 which, according to the free radical pecking order proposed by Buettner,59 is capable of abstracting allylic and bisallylic hydrogens on thermodynamical grounds. However, direct interaction with oleate derivatives was not observed,60 although a similar reaction (i.e., LOO•-mediated hydrogen abstraction) is suggested to propagate autoxidation of oleic acid.61 On the other hand, the interaction of polyunsaturated fatty acid derivatives with •OOH has been demonstrated and rate constants for hydrogen abstraction were proportional to the number of bisallylic positions in the olefinic chain.60 Still, the reported bimolecular rate constant k ∼ 1.2 × 103 L mol-1 s-1 for the abstraction of linolenic hydrogens is a 1000-fold smaller than the experimentally

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SCHEME 3: Radical Formation in a Light-Exposed Solution Containing Lumiflavin (LF) and Methyl Linoleate, As Determined by Spin Trapping Experimentsa

a

Observed spin adducts (with varying spin traps) are depicted in boxes.

TABLE 4: Spin Adducts, Detected after Irradiation of Various Fatty Acid Methyl Esters (∼200 mM) in the Presence of Lumiflavin (25 µM) and DMPIO (5 mM)

Figure 5. (A) EPR signal observed during 440 nm irradiation of a nitrogen-purged mixture of lumiflavin and DMPIO in acetonitrile. (B) Experimental (upper) and simulated (lower) EPR signal corresponding to DMPIO adducts detected after irradiation (5 min) of lumiflavin in the presence of 200 mM methyl linoleate. (C) Analogous to panel B, but methyl linolenate is substituted for methyl linoleate. Vertical arbitrary scale indicates intensities of the respective signals.

determined rate constant for quenching of triplet-excited flavins, supporting the importance of initiation via type I photooxidation. Spin Trapping by 2,2-Dimethyl-4-phenyl-2H-imidazole 1-oxide (DMPIO). Because of the formation and significant build-up of i, analysis of other DMPO adducts is severely complicated. Therefore, DMPIO, a lipophilic nitrone that fails to form stable adducts with superoxide62 was substituted for DMPO in the reaction mixture. Thus, no spin adduct was detected on irradiation of a lumiflavin solution (Figure 5A), which corroborates the identification of i in the DMPO trapping experiments. Addition of methyl linoleate followed by lightexposure readily produced an intense signal (Figure 5B). An identical, but more intense spin pattern was observed with methyl linolenate as substrate (Figure 5C), a consequence of the doubling of the oxidation sites (1 vs 2). The coupling constant with the nitroxyl nitrogen, which largely depends on the electronic nature of the incipient radical center, suggested trapping an alkoxyl radical.62,63 The different value for the β-hydrogen in the observed LO•-DMPIO compared to the

compound

RAa

aNb

aHc

SAd

methyl oleate methyl linoleate methyl linolenate

100 100 100

12.3 12.3 12.3

14.2 14.4 14.4

xiv

a Relative abundance (%). b Coupling constant to DMPIO nitrogen (Gauss). c Coupling constant to DMPIO β-hydrogen (Gauss). d Spin adduct identification (Scheme 3).

reported RO•-DMPIO adduct63 (14.4 G vs 12.5 G) was likely caused by the difference in conformation due to steric influence of the carbon chain (Table 4). Spin Trapping by 2-Methyl-2-nitrosopropane (MNP). Unlike nitrone spin traps DMPO and DMPIO, MNP adds radicals directly to the nitroso nitrogen. As a consequence, addition of carbon-centered radicals is faster than to DMPO64 and oxygen-centered radicals are discriminated due to the instability of the resulting tBu-N(O•)-OR adducts. Accordingly, MNP was implemented in an attempt to detect initial carboncentered lipid-like radicals, L•, which were suspected to be involved in the early events of the photooxidation reaction. In this respect, it is worth noting that MNP offers considerably more structural information than its nitrone counterparts, as multiplicity of the resulting spin pattern is directly related to the number of hydrogens prevailing on the incipient radical center. Trapping of carbon-centered radicals was readily observed after light-exposure of a mixture containing methyl linoleate (200 mM), lumiflavin (25 µM), and MNP (10 mM) (Figure 6A). Unraveling the tangle of different spin adducts was laborious, but one spin adduct was immediately identified due to its resemblance to the single radical species trapped during irradiation of methyl oleate (Figure 6B). Simulation revealed a

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Huvaere et al.

Figure 6. Experimental (upper) and simulated (lower) traces of EPR signals of MNP adducts detected during irradiation of lumiflavin in the absence and presence of various fatty acid methyl esters. (A) 200 mM methyl linoleate; (B) 200 mM methyl oleate; (C) analogous to panel B, but after prolonged irradiation (>15 min); (D) no substrate added, but lumiflavin concentration was increased (250 µM instead of 25 µM). Vertical arbitrary scale indicates intensities of the respective signals.

TABLE 5: Spin Adducts, Detected after 5 Minutes of Irradiation (Unless Stated Otherwise) of Various Fatty Acid Methyl Esters (∼200 mM) in the Presence of Lumiflavin (25 µM) and MNP (10 mM) compound no substrate methyl oleate methyl oleate (∼ 10 min) methyl linoleate

methyl linolenate

RAa 100 100 55 45 41 36 12 8 3 65 25 10

aNb 15.7 8.0 8.0 15.7 18.0 14.4 15.5 18.0 8.0 14.8 15.4 18.2

aHc

SAd

e

iv

nd nd nd nd 1.7, 1.7 1.9 nd 3.4 nd 1.9 nd 1.6, 2.0

iv xv xvi iv xvii xviii iv

a Relative abundance (%). b Coupling constant to MNP nitrogen (Gauss). c Coupling constant to substrate hydrogen(s) (Gauss). d Spin adduct identification (Schemes 2 and 3). e nd: not detected.

Figure 7. Structures of methyl stearate and oleyl alcohol.

characteristic, small coupling to the nitroxyl nitrogen (aN ∼ 8.0 G) which, in combination with the lack of hydrogen coupling, typically referred to addition of an acyl radical (Table 5).65 A quasi-identical acyl-MNP adduct was found in the investigation of catalytic peroxidation of linoleic acid by manganesedependent peroxidase,66 but mechanistic details were not disclosed. Its formation in the irradiation experiments was initially suspected to involve ester disintegration by addition of superoxide, which has a nucleophilicity that is superior to methoxide.67 A similar mechanism was previously suggested,68,69 but failure to detect acyl radicals in a light-exposed mixture containing methyl stearate (Figure 7) refuted the feasibility of such pathway. Instead, an acyl-MNP adduct was observed after substituting oleyl alcohol for methyl stearate, suggesting a crucial role for the double bond. In this respect fatty acid

hydroperoxides, resulting from peroxidation at the (bis)allylic position, were shown to generate acyl radicals (detected as DMPO adduct) after exposure to catalytic lipoxygenase or cytochrome P-450 activity.49,53,70 Decomposition of the hydroperoxide to the corresponding alkoxyl radical LO• was pivotal herein, furnishing an identical intermediate as observed in our irradiation experiments. Similar follow-up radical reactions were thus expected,71 including a β-scission pathway with formation of an aldehyde and a reactive, primary alkyl radical (similar as x in the degradation pathway of methyl linoleate depicted in Scheme 3). Subsequent hydrogen abstraction from the aldehyde moiety corroborates detection of a spin adduct with an acyl radical (analogous to xviii derived from methyl linoleate oxidation), but homolytic cleavage of the C(O)-H bond by alkyl radicals is relatively inefficient. An estimated rate constant of 5.0 × 103 L mol-1 s-1 at 75 °C has been proposed,72 which implies that alkoxyl radicals (such as LO•) are considerably more reactive toward abstraction of the aldehyde hydrogen (k ∼ 7.0 × 107 L mol-1 s-1 for reaction with tBuO• in benzene at 0 °C).73 Peroxyl radicals (LOO•) are probably not involved in acyl radical formation as a result of their slow interaction with aldehydes (∼ 4.8 L mol-1 s-1 in heptane at 0 °C).74,75 Because of interference of multiple degradation processes, the acyl adduct prevailed only as minor signal in the methyl linoleate spectrum (Table 5). One of the major species is an MNP adduct with coupling to two identical hydrogens (aN ∼ 18.0 G and 2 × aH ∼ 1.7 G) that was immediately associated with trapping of a primary alkyl radical (possibly x resulting from the β-scission pathway). The second major species (xvi; aN ∼ 14.4 G and aH ∼ 1.9 G) was very similar to the previously identified allylic free radical adduct in lipoxygenase-catalyzed oxidation of linoleic acid.76 However, care should be taken when attributing this species directly to flavin-mediated oxidation, as in our experiments a similar adduct was detected in a mixture of MNP and methyl linoleate stored in the dark. Indeed, nitroso compounds are known to undergo a thermal ene reaction with olefins,77 producing a nitroxyl radical that possibly misleads the observer in identifying radical processes.78–80 Mixtures of substrate and lumiflavin were therefore purged with nitrogen in the absence of MNP, then the latter was added just prior to irradiation and concurrent EPR analysis. Since the thermal addition reaction is too slow to be relevant in the time frame of the irradiation experiments (