Kevin Huvaere

Light-Induced Oxidation of Tryptophan and Histidine. Reactivity of Aromatic N-Heterocycles toward Triplet-Excited Flavins

Mechanisms of flavin-mediated photooxidation of electron-rich amino acids tryptophan and histidine were investigated for aqueous solutions. Indole, representing the tryptophan side chain in proteins, reacted at nearly diffusion controlled rates (k approximately 2.7 x 10(9) L mol(-1) s(-1) at 293 K) with the triplet-excited flavin state, but reactions of imidazole (and histidine) were significantly slower (k < 2.0 x 10(8) L mol(-1) s(-1)) as determined by laser flash photolysis. Oxidation rates of derivates were invariably susceptible to electronic factors affecting incipient radical cation stability, while no primary kinetic hydrogen/deuterium isotope effect was observed for imidazole. Thus reaction by electron transfer was proposed in contrast to a direct hydrogen abstraction. Unlike indole compounds, imidazole derivatives suffered from the presence of a basic imino nitrogen (=N-), which caused the rate constant of histidine free base (k approximately 1.8 x 10(8) L mol(-1) s(-1)) to drop considerably upon protonation. Complexation of the imino nitrogen with transition metals provoked changes in reactivity, as rate constants decreased after addition of Zn(2+) (k of 4-methylimidazole, as histidine model, decreased from 9.0 x 10(8) L mol(-1) s(-1) in the absence to 4.1 x 10(8) L mol(-1) s(-1) in the presence of ZnCl(2)). The pyrrole nitrogen (-NH-) was not directly involved in complexation reactions, but its electron density increased upon interaction with hydrogen bond-accepting anions and resulted in higher rate constants (k of 4-methylimidazole increased from 9.0 x 10(8) L mol(-1) s(-1) to 2.0 x 10(9) L mol(-1) s(-1) after addition of NaOAc). The high rate constants were in agreement with a large thermodynamical driving force, as calculated from oxidation peak potentials determined electrochemically. After oxidation, resulting radical cations were readily deprotonated and trapped by 2-methyl-2-nitrosopropane, as detected by electron paramagnetic resonance spectroscopy. Indole-derived spin adducts were attributed to selective trapping of C(3)-centered radicals, whereas spin adducts with imidazole-derivatives arose from both carbon and nitrogen-centered imidazolyl radicals.

Light-induced oxidation of unsaturated lipids as sensitized by flavins.

Triplet-excited riboflavin ((3)RF*) 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 approximately 3.0 x 10(5) L mol(-1) s(-1) at 25 degrees C for methyl linoleate and 3.1 x 10(6) L mol(-1) s(-1), with DeltaH(double dagger) = 22.6 kJ mol(-1) and DeltaS(double dagger) = -62.3 J K(-1) mol(-1), for methyl linolenate in acetonitrile/water (8:2, v/v). For methyl oleate, k was <10(4) L mol(-1) s(-1). For comparison, beta-casein was found to have a rate constant k approximately 4.9 x 10(8) L mol(-1) s(-1). Singlet-excited flavin was not quenched by the esters as evidenced by insensitivity of steady-state fluorescence to their presence. Density functional theory (DFT) calculations showed that electron transfer from unsaturated fatty acid esters to triplet-excited flavins is endergonic, while a formal hydrogen atom transfer is exergonic (DeltaG(o)(HAT) = -114.3, -151.2, and -151.2 kJ mol(-1) for oleate, linoleate, and linolenate, respectively, in acetonitrile). The reaction is driven by acidity of the lipid cation radical for which a pK(a) approximately -0.12 was estimated by DFT calculations. Absence of electrochemical activity in acetonitrile during cyclic voltammetry up to 2.0 V versus NHE confirmed that DeltaG(o)(ET) > 0 for electron transfer. Interaction of methyl esters with (3)RF* 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 (3)RF* 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 10(4)), 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.

Flavin -induced photodecomposition of sulfur-containing amino acids is decisive in the formation of beer lightstruck flavor

Photooxidation of sulfur-containing amino acids and derivatives readily occurs upon visible-light irradiation in the presence of flavins. The sulfur moiety seems pivotal for interaction, as was determined from kinetic analyses using laser flash photolysis spectroscopy. After photooxidation, the resulting radical intermediates were characterized by addition to a spin trap, followed by electron paramagnetic resonance spectroscopy and evaluation of the coupling constants. The presence of the proposed radical intermediates was strongly supported by the identification of the reaction products using mass spectrometry. Accordingly, feasible degradation pathways for various sulfur-containing amino acids and derivatives were proposed. It was finally proven that flavin-induced photoproduction of sulfhydryl radicals and recombination with a 3-methylbut-2-enyl radical, derived from the photodegradation of hop-derived isohumulones, are decisive in the formation of beer lightstruck flavor.

Riboflavin-sensitized photooxidation of isohumulones and derivatives

Isohumulones, the bitter principles in beer, are decomposed by light-induced reactions, thereby adversely affecting beer quality. Upon absorption of visible light, riboflavin is excited and interacts with isohumulones, as well as with oxidized and reduced derivatives thereof. Reaction kinetics were investigated by laser flash photolysis at 355 nm and at 440 nm, and analysis of kinetic data afforded detailed insights into the reaction mechanism.

Antioxidant properties of green tea extract protect reduced fat soft cheese against oxidation induced by light exposure.

The effect of two different antioxidants, EDTA and green tea extract (GTE), used individually or in combination, on the light-induced oxidation of reduced fat soft cheeses (0.2 and 6% fat) was investigated. In samples with 0.2% fat, lipid hydroperoxides as primary lipid oxidation products were not detected, but their interference was suggested from the formation of secondary lipid oxidation products such as hexanal and heptanal. The occurrence of these oxidation markers was inhibited by spiking with 50 ppm EDTA or 750 ppm GTE, or a combination of the two prior to irradiation. In contrast, addition of 50 ppm EDTA to samples with 6% fat was ineffective, but 750 ppm GTE (alone or in combination with EDTA) strongly reduced levels of hexanal and heptanal. Accumulation of primary lipid hydroperoxides was not affected by GTE, hence antioxidative activity was ascribed to scavenging of hexanal and heptanal precursors. These radical intermediates result from hydroperoxide disintegration, and subsequent scavenging by GTE, which acts as a radical sink, corroborates the intense signal observed by electron paramagnetic resonance (EPR) spectroscopy.

Photooxidative degradation of beer bittering principles: product analysis with respect to lightstruck flavour formation

Isohumulones, the main bittering agents in beer, are decomposed by light-induced reactions, thereby leading to radical precursors on the pathway to lightstruck flavour formation. Excited flavins, formed on visible-light irradiation, readily interact with isohumulones, as well as with reduced and oxidized derivatives thereof. From identification of both volatile and non-volatile reaction products thus formed, feasible degradation mechanisms are proposed.

Photolysis of hop-derived trans-iso-α-acids and trans-tetrahydroiso-α-acids: product identification in relation to the lightstruck flavour of beer

The photolysis of hop-derived trans-iso-alpha-acids (2a-c; naturally occurring bitter compounds present in beer) and of trans-tetrahydroiso-alpha-acids (5a-c; semi-synthetic advanced hop products) was investigated at 300 nm in methanol. The complex photoreaction mixtures were separated by high-performance liquid chromatography (HPLC) using diode array detection and the major photoreaction products were identified by HPLC-mass spectroscopy. The main part of the mixture consisted of compounds, which originated from recombination of radicals derived from Norrish Type I photocleavage of the acyloin moiety in both trans-iso-alpha-acids and trans-tetrahydroiso-alpha-acids. The results confirm the intermediacy of radicals that were previously identified by time-resolved electron paramagnetic resonance and they bear relevance to the formation of the lightstruck flavour that is generated when beer is exposed to light. Additionally, new photoproducts were found that are formed by photochemical reactions hitherto undiscovered in hop chemistry, including photoenolization of trans-isohumulone (2a) leading to trans-alloisohumulone (13a) and a retro oxa-di-pi-methane rearrangement in trans-isohumulone (2a) and trans-tetrahydroiso-alpha-acids to humulone (1a) and tetrahydro-alpha-acids (23a-b), respectively.

Quenching of triplet-excited flavins by flavonoids. Structural assessment of antioxidative activity.

The mechanism of flavin-mediated photooxidation of flavonoids was investigated for aqueous solutions. Interaction of triplet-excited flavin mononucleotide with phenols, as determined by laser flash photolysis, occurred at nearly diffusion-controlled rates (k approximately 1.6 x 10(9) L mol(-1) s(-1) for phenol at pH 7, 293 K), but protection of the phenolic function by methylation inhibited reaction. Still, electron transfer was proposed as the dominating mechanism due to the lack of primary kinetic hydrogen/deuterium isotope effect and the low activation enthalpy (<20 kJ mol(-1)) for photooxidation. Activation entropy worked compensating in a series of phenolic derivatives, supporting a common oxidation mechanism. An ortho-hydroxymethoxy pattern was equally reactive (k approximately 2.3 x 10(9) L mol(-1) s(-1) for guaiacol at pH 7) as compounds with ortho-dihydroxy substitution (k approximately 2.4 x 10(9) L mol(-1) s(-1) for catechol at pH 7), which are generally referred to as good antioxidants. This refutes the common belief that stabilization of incipient phenoxyl radicals through intramolecular hydrogen bonding is the driving force behind the reducing activity of catechol-like compounds. Instead, such bonding improves ionization characteristics of the substrates, hence the differences in reactivity with (photo)oxidation of isolated phenols. Despite the similar reactivity, radicals from ortho-dihydroxy compounds are detected in high steady-state concentrations by electron paramagnetic resonance (EPR) spectroscopy, while those resulting from oxidation of ortho-hydroxymethoxy (or isolated phenolic) patterns were too reactive to be observed. The ability to deprotonate and form the corresponding radical anions at neutral pH was proposed as the decisive factor for stabilization and, consequently, for antioxidative action. Thus, substituting other ionizable functions for the ortho- or para-hydroxyl in phenolic compounds resulted in stable radical anion formation, as demonstrated for para-hydroxybenzoic acid, in contrast to its methyl ester.

Characterization of Peroxides Formed by Riboflavin and Light Exposure of Milk. Detection of Urate Hydroperoxide as a Novel Oxidation Product

Characterization of peroxides by size exclusion chromatography (SEC) of milk following exposure to riboflavin and light showed that hydrogen peroxide was the most abundant peroxide formed since it could be removed by catalase. Formation of peroxides after separation by SEC showed that hydrogen peroxide formation was primarily increased in the presence of caseins and ascorbate, although whey proteins also were found to contribute. Caseins and beta-lactoglobulin also formed catalase-resistant peroxides, presumably protein hydroperoxides. A catalase-resistant and unstable peroxide was observed in fractions containing urate. Experiments performed with pure urate suggested that urate radicals reacted further with superoxide leading to a urate hydroperoxide. Electron paramagnetic resonance spectroscopy using spin-traps showed that the presence of oxygen was required for urate radical formation, which could be assigned as nitrogen-centered radicals. These results suggest a new route during light-induced oxidation sensitized by flavins, in effect making urate pro-oxidative.

Flavonoid deactivation of excited state flavins: reaction monitoring by mass spectrometry.

Flavin mononucleotide (FMN, as a B(2) vitamin model) was shown to induce dimerization of flavonoids (flavanone, apigenin, naringenin, eriodictyol, taxifolin, catechin, kaempferol, luteolin, quercetin, rutin, and seven smaller model phenols studied) as the major photoreaction, when aqueous solutions were exposed to visible light using a new, real-time electrospray ionization mass-spectrometric (ESI-MS) technique supported by LC-MS and MS(2) analysis. Electrophilic intermediates such as transient radical cations, o-quinones, and p-quinone methide were proposed to be involved in the coupling process. The C(3)-OH in flavon-3-ols gave rise to atypical compounds such as a depside or a dioxane-linked dimer. Flavonoid dimers, formed in vegetal extracts added to food during storage in light and for which structures are proprosed based on MS and MS(2), may affect colloidal stability, color, astringency, and antioxidative capacity.