Selectivity of Dietary Phenolics for Inhibition of Human Monoamine Oxidases A and B.

Monoamine oxidases (MAOs) regulate local levels of neurotransmitters such as dopamine, norepinephrine, and serotonin and thus have been targeted by drugs for the treatment of certain CNS disorders. However, recent studies have shown that these enzymes are upregulated with age in nervous and cardiac tissues and may be involved in degeneration of these tissues, since their metabolic mechanism releases hydrogen peroxide leading to oxidative stress. Thus, targeting these enzymes may be a potential anti-aging strategy. The purpose of this study was to compare the MAO inhibition and selectivity of selected dietary phenolic compounds, using a previously validated assay that would avoid interference from the compounds. Kynuramine metabolism by human recombinant MAO-A and MAO-B leads to formation of 4-hydroxyquinoline, with Vmax values of 10.2±0.2 and 7.35±0.69 nmol/mg/min, respectively, and Km values of 23.1±0.8 µM and 18.0±2.3 µM, respectively. For oral dosing and interactions with the gastrointestinal tract, curcumin, guaiacol, isoeugenol, pterostilbene, resveratrol, and zingerone were tested at their highest expected luminal concentrations from an oral dose. Each of these significantly inhibited both enzymes except for zingerone, which only inhibited MAO-A. The IC50 values were determined, and selectivity indices (MAO-A/MAO-B IC50 ratios) were calculated. Resveratrol and isoeugenol were selective for MAO-A, with IC50 values of 0.313±0.008 and 3.72±0.20 µM and selectivity indices of 50.5 and 27.4, respectively. Pterostilbene was selective for MAO-B, with IC50 of 0.138±0.013 µM and selectivity index of 0.0103. The inhibition of resveratrol (MAO-A) and pterostilbene (MAO-B) was consistent with competitive time-independent mechanisms. Resveratrol 4'-glucoside was the only compound which inhibited MAO-A, but itself, resveratrol 3-glucoside, and pterostilbene 4'-glucoside failed to inhibit MAO-B. Additional studies are needed to establish the effects of these compounds on MAO-A and/or MAO-B in humans.

Enhanced production of camptothecin and biological preparation of N 1-acetylkynuramine in Camptotheca acuminata cell suspension cultures.

The Camptotheca acuminata cell suspension cultures were established to produce the well-known antitumor monoterpene indole alkaloid camptothecin (CAM). Most CAM was present in the broth of the C. acuminata cell suspension cultures. The CAM production was evidenced to be attenuated when the C. acuminata cell suspension cultures were continuously subcultured and grown under identical axenic conditions. A practical cryopreservation and recovery procedure was established to maintain the C. acuminata cell suspension cultures. Biotic and abiotic elicitors were administrated to the C. acuminata cell suspension cultures to restore and enhance CAM production. Of them, sorbitol, a well-known hyperosmotic stressor, was proven to be the most effective elicitor that stimulates a ∼500-fold increase of CAM production. The committed biosynthetic precursors of CAM, tryptamine and secologanin, were feed to the C. acuminata cell suspension cultures and the CAM production is not remarkably increased. However, N 1-acetylkynuramine (NAK), an important metabolite of kynuramine pathway, was isolated and identified from the cell suspension cultures feeding with tryptamine. The present work provides an efficient method to produce CAM and NAK using the C. acuminata cell suspension cultures. The biotransformation of tryptamine to NAK sheds lights on the biosynthetic formation of the pyrroloquinoline moiety of CAM.

Development of urea and thiourea kynurenamine derivatives: synthesis, molecular modeling, and biological evaluation as nitric oxide synthase inhibitors.

Herein we describe the synthesis of a new family of kynurenamine derivatives with a urea or thiourea moiety, together with their in vitro biological evaluation as inhibitors of both neuronal and inducible nitric oxide synthases (nNOS and iNOS, respectively), enzymes responsible for the biogenesis of NO. These compounds were synthesized from a 5-substituted-2-nitrophenyl vinyl ketone scaffold in a five-step procedure with moderate to high chemical yields. In general, the assayed compounds show greater inhibition of iNOS than of nNOS, with 1-[3-(2-amino-5-chlorophenyl)-3-oxopropyl]-3-ethylurea (compound 5 n) being the most potent iNOS inhibitor in the series and the most iNOS/nNOS-selective compound. In this regard, we performed molecular modeling studies to propose a binding mode for this family of compounds to both enzymes and, thereby, to elucidate the differential molecular features that could explain the observed selectivity between iNOS and nNOS.

Profiling substrate specificity of two series of phenethylamine analogs at monoamine oxidase A and B.

The membrane bound enzyme monoamine oxidase exist in two splice variants designated A and B (MAO-A and MAO-B) and are key players in the oxidative metabolism of monoamines in mammalians. Despite their importance and being a prevalent target for the development of inhibitors as drugs, no systematic study of substrate specificity has been reported. In this study we present a systematic study of the MAO-A and MAO-B substrate specificity profile by probing two series of phenethylamine analogs. Km and kcat values were determined for four N-alkyl analogs 2-5 and four aryl halide analogs 6-9 at MAO-A and MAO-B. A following in silico study disclosed a new adjacent compartment to the MAO-B substrate pocket defined by amino acids Tyr188, Tyr435, Tyr398, Thr399, Cys172 and Gly434. This new insight is important for the understanding of the substrate specificity of the MAO-B enzyme and will be relevant for future drug design within the field of monoamines.

Substituents effects on activity of kynureninase from Homo sapiens and Pseudomonas fluorescens.

A series of substituted kynurenines (3-bromo-DL, 3-chloro-DL, 3-fluoro-DL, 3-methyl-DL, 5-bromo-L, 5-chloro-L, 3,5-dibromo-L and 5-bromo-3-chloro-DL) have been synthesized and tested for their substrate activity with human and Pseudomonas fluorescens kynureninase. All of the substituted kynurenines examined have substrate activity with both human as well as P. fluorescens kynureninase. For the human enzyme, 3- and 5-substituted kynurenines have kcat and kcat/Km values higher than L-kynurenine, but less than that of the physiological substrate, 3-hydroxykynurenine. However, 3,5-dibromo- and 5-bromo-3-chlorokynurenine have kcat and kcat/Km values close to that of 3-hydroxykynurenine with human kynureninase. The effects of the 3-halo substituents on the reactivity with human kynureninase may be due to electronic effects and/or halogen bonding. In contrast, for the bacterial enzyme, 3-methyl, 3-halo and 3,5-dihalokynurenines are much poorer substrates, while 3-fluoro, 5-bromo, and 5-chlorokynurenine have kcat and kcat/Km values comparable to that of its physiological substrate, L-kynurenine. Thus, 5-bromo and 5-chloro-L-kynurenine are good substrates for both human as well as bacterial enzyme, indicating that both enzymes have space for substituents in the active site near C-5. The increased activity of the 5-halokynurenines may be due to van der Waals contacts or hydrophobic effects. These results may be useful in the design of potent and/or selective inhibitors of human and bacterial kynureninase.

On the free radical scavenging activities of melatonin's metabolites, AFMK and AMK.

The reactions of N(1) -acetyl-N(2) -formyl-5-methoxykynuramine (AFMK) and N(1) -acetyl-5-methoxykynuramine (AMK) with (•) OH, (•) OOH, and •OOCCl3 radicals have been studied using the density functional theory. Three mechanisms of reaction have been considered: radical adduct formation (RAF), hydrogen transfer (HT), and single electron transfer (SET). Their relative importance for the free radical scavenging activity of AFMK and AMK has been assessed. It was found that AFMK and AMK react with •OH at diffusion-limited rates, regardless of the polarity of the environment, which supports their excellent •OH radical scavenging activity. Both compounds were found to be also very efficient for scavenging •OOCCl3 , but rather ineffective for scavenging •OOH. Regarding their relative activity, it was found that AFMK systematically is a poorer scavenger than AMK and melatonin. In aqueous solution, AMK was found to react faster than melatonin with all the studied free radicals, while in nonpolar environments, the relative efficiency of AMK and melatonin as free radical scavengers depends on the radical with which they are reacting. Under such conditions, melatonin is predicted to be a better •OOH and •OOCCl3 scavenger than AMK, while AMK is predicted to be slightly better than melatonin for scavenging •OH. Accordingly it seems that melatonin and its metabolite AMK constitute an efficient team of scavengers able of deactivating a wide variety of reactive oxygen species, under different conditions. Thus, the presented results support the continuous protection exerted by melatonin, through the free radical scavenging cascade.

Proteoliposome-based capillary electrophoresis for screening membrane protein inhibitors.

A method for screening of monoamine oxidase (MAO) inhibitor was carried out using capillary electrophoresis (CE) based on the interaction of MAO and its substrate kynuramine (Kyn). Bioactive proteoliposome was reconstituted by liposome and MAO and then was applied as the pseudostationary phase (PSP) of CE to mimic the interaction between the enzyme and its substrate. N-prolmrgyl-R-2-heptylamine (R-2-HPA) and rasagiline [N-propargyl-1-(R)-aminoindan], which are two kinds of MAO inhibitors, were added into the running buffers containing proteoliposome. The results showed that the relative migration time ratio (RMTR × 10(-1)) values of Kyn were enhanced from 8.88 to 9.31 with an increase of the concentrations of rasagiline from 10(-6) to 1 mM. However, the RMTR values of Kyn were enhanced from 8.83 to 9.14 with an increase of the concentrations of R-2-HPA from 10(-6) to 1 mM. The RMTR value of Kyn in the presence of rasagiline was larger than that in the presence of R-2-HPA when rasagiline and R-2-HPA were at the same concentration. The results indicated that the interaction between Kyn and MAO was weakened with the increase of the inhibitors. In addition, the results of offline incubation showed that the inhibitions of rasagiline were 100.0, 72.1, 51.8 and 5.4% at the concentration of 1, 10(-2), 10(-4) and 10(-6) mM; moreover, the inhibitions of R-2-HPA were 70.0, 44.9, 4.1 and 0.9% at the concentrations of 1, 10(-2), 10(-4) and 10(-6) mM. The inhibition efficiency of rasagiline was stronger than that of R-2-HPA at the same concentration. Additionally, the interaction between Kyn and liposome was also investigated. This newly developed method might provide a potential tool for screening MAO inhibitor.

Oxidation of melatonin by taurine chloramine.

Melatonin is widely known for its antioxidant, immunomodulatory, and anti-inflammatory effects. Hypochlorous acid (HOCl) is one example of an endogenous oxidant that is promptly neutralized by melatonin. Melatonin also inhibits myeloperoxidase, the enzyme that catalyzes the oxidation of chloride to HOCl. Taurine is the most abundant free amino acid in leukocytes. In activated neutrophils, taurine is converted to taurine chloramine (Tau-NHCl) through a reaction with HOCl. In addition, the related compound taurine bromamine (Tau-NHBr) can be released by neutrophils and eosinophils. The aim of this study was to investigate the reactivity of Tau-NHCl and Tau-NHBr with melatonin. We found that melatonin can react with either Tau-NHCl or Tau-NHBr, leading to the production of 2-hydroxymelatonin and N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK). The reaction was pH-dependent, and it occurs more rapidly at a slightly acidic pH. Tau-NHBr was significantly more reactive than Tau-NHCl. Using Tau-NHBr as the oxidizing agent, 1 mm melatonin was oxidized in less than 1 min. The pH dependence of the reaction with Tau-NHCl and the increased reactivity of Tau-NHBr can be explained by a mechanism based on the initial attack of chloronium (Cl(+)) or bromonium (Br(+)) ions on melatonin. We also found that the addition of iodide to the reaction medium increased the yield of AFMK. These findings could contribute to the establishment of new functions for melatonin in inflammatory and parasitic diseases, where the role of this indoleamine has been extensively investigated.

Analysis of N1-acetyl-N2-formyl-5-methoxykynuramine/N1-acetyl-5-methoxy-kynuramine formation from melatonin in mice.

The interactions of melatonin, a potent endogenous antioxidant, with reactive oxygen species generate several products that include N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) and N(1)-acetyl-5-methoxy-kynuramine (AMK). The physiological or pathological significance of AFMK/AMK formation during the process of melatonin metabolism in mammals has not been clarified. Using a metabolomic approach in the current study, the AFMK/AMK pathway was thoroughly investigated both in mice and humans. Unexpectedly, AFMK and AMK were not identified in the urine of humans nor in the urine, feces or tissues (including liver, brain, and eyes) in mice under the current experimental conditions. Metabolomic analysis did identify novel metabolites of AMK, i.e. hydroxy-AMK and glucuronide-conjugated hydroxy-AMK. These two newly identified metabolites were, however, not found in the urine of humans. In addition, oxidative stress induced by acetaminophen in the mouse model did not boost AFMK/AMK formation. These data suggest that AFMK/AMK formation is not a significant pathway of melatonin disposition in mice, even under conditions of oxidative stress.

Reactions of the melatonin metabolite N(1)-acetyl-5-methoxykynuramine with carbamoyl phosphate and related compounds.

N-[2-(6-methoxyquinazolin-4-yl)-ethyl] acetamide (MQA) is a compound formed from the melatonin metabolite N(1)-acetyl-5-methoxykynuramine (AMK). We followed MQA production in reaction systems containing various putative reaction partners, in the absence and presence of hydrogen peroxide and/or copper(II). Although MQA may be formally described as a condensation product of either N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) with ammonia, or AMK with formamide, none of these combinations led to substantial quantities of MQA. However, MQA formation was observed in mixtures containing AMK, hydrogen peroxide, hydrogen carbonate and ammonia, or AMK, hydrogen peroxide, copper(II) and potentially carbamoylating agents, such as potassium cyanate or, more efficiently, carbamoyl phosphate. In the presence of hydrogen peroxide, copper(II) and carbamoyl phosphate, MQA was the major product obtained from AMK, but the omission of copper(II) mainly led to another metabolite, 3-acetamidomethyl-6-methoxycinnolinone (AMMC). This was caused by nitric oxide (NO) generated under oxidative conditions from carbamoyl phosphate, as shown by an NO spin trap. MQA formation with carbamoyl phosphate was not due to the possible decomposition product, formamide. The reaction of AMK with carbamoyl phosphate under oxidative conditions, in which inorganic phosphate and water are released and which differs from the typical process of carbamoylation via isocyanate, may be considered as a new physiological route of MQA formation.

Online H/D exchange liquid chromatography as a support for the mass spectrometric identification of the oxidation products of melatonin.

The hydrogen-deuterium exchange of protonated melatonin and its in vitro oxidation end-products have been examined by liquid chromatography coupled with ion-trap mass spectrometry. Specific H/D scrambling of protons in the C2 and C4 positions of the indole ring during gas-phase fragmentation process was observed for both melatonin and its oxidation products. Collision-induced dissociation spectra showed losses of variably deuterated NH(3), H(2)O and CH(3)CONH(2). In addition, a similar H/D scrambling behaviour was observed for the oxidation products, obtained from the opening of the indole ring by oxidative attack. Fragmentation pathways are proposed and H/D scrambling has been employed as a fingerprint, allowing identification of N(1)-acetyl-5-methoxykynurenin (AMK), N(1)-acetyl-N(2)-formyl-5-methoxykynurenin (AFMK), dehydro-AFMK and hydroxymelatonin as the oxidation products of melatonin in vitro.

The melatonin metabolite N-acetyl-5-methoxykynuramine is a potent singlet oxygen scavenger.

Singlet oxygen was generated by means of rose bengal under irradiation by visible light. N(1)-acetyl-5-methoxykynuramine (AMK) was rapidly destroyed by this reactive oxygen species, whereas its formylated precursor, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), was remarkably inert. At photon fluence rates of 1400 mumol photons/m(2)s, and using 20 mum rose bengal, most of initially 0.2 mm AMK was destroyed within 2 min, whereas AFMK remained practically unchanged for much longer periods of time. Competition experiments with other scavengers revealed the following order of reactivity towards singlet oxygen: diazabicyclo-[2,2,2]-octane (DABCO) << imidazole < 4-ethylphenol < N(alpha)-acetylhistidine < histidine < melatonin < AMK, the last one being about 150 times more effective than DABCO. Contrary to the oxidation in free radical-generating systems, AMK did not form adducts with the tyrosine side chain fragment, 4-ethylphenol, under the influence of singlet oxygen. In UV-exposed cells (keratinocytes, plant cells) it is likely to be more rapidly destroyed by singlet oxygen than formed from AFMK.

MT3/QR2 melatonin binding site does not use melatonin as a substrate or a co-substrate.

Quinone reductase 2 (QR2, E.C. 1.10.99.2) is implicated in cell reactive oxygen species production. The catalytic activity of this enzyme is inhibited by 1 microM of melatonin. QR2 was identified as the third melatonin binding site (MT3). It is of major importance to understand the exact roles of melatonin and QR2 in oxidative stress. A fascinating possibility that melatonin could serve as a co-substrate or substrate of QR2 was hypothesized recently. In the current investigation, nuclear magnetic resonance studies of the QR2 catalytic reaction were performed, the results led us to conclude that, whatever the conditions, melatonin is not cleaved off to form N1-acetyl-N2-formyl-5-methoxykynurenine by a catalytically active QR2, very strongly indicating that melatonin is neither a substrate nor a co-substrate of this enzyme. Further studies are needed in order to better understand the relationship between MT3/QR2, melatonin and redox status of the cells, in order to better explain the anti-oxidant activities of melatonin at pharmacological concentrations (>1 microM).

Novel pathway for N1-acetyl-5-methoxykynuramine: UVB-induced liberation of carbon monoxide from precursor N1-acetyl-N2-formyl-5-methoxykynuramine.

Irradiation of the melatonin metabolite N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) with UV light of 254 nm causes the release of carbon monoxide (CO) and, thus, deformylation to N(1)-acetyl-5-methoxykynuramine (AMK). Liberation of CO was demonstrated by reduction of PdCl(2) to metallic palladium, under avoidance of actions by other reductants. Photochemical AMK formation was not due to UV-induced hydroxyl radicals, because the reaction also took place with high efficiency in ethanol and 2-propanol. Moreover, AMK was generated from AFMK by UVB on a dry thin layer chromatographic plate. Although AMK seems to be the major primary product generated by UVB radiation, prolonged exposure of AFMK led to various other products, especially formed by destruction of AMK, as shown by irradiation of this latter compound. With regard to the demonstration of melatonin in skin and substantial amounts of AFMK in keratinocytes, these findings may be of dermatologic relevance.

Reactions of the NO redox forms NO+, *NO and HNO (protonated NO-) with the melatonin metabolite N1-acetyl-5-methoxykynuramine.

The different NO redox forms, NO+, *NO and HNO (=protonated NO-), were compared for their capabilities of interacting with the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK), using NO+SbF6-, PAPA-NONOate and Angeli's salt as donors of the respective NO species. Particular attention was paid to stability and possible interconversions of the redox forms. *NO formation was followed by measuring the decolorization of 2-(trimethylammonio-phenyl)-4,4,5,5-tetramethyl-imidazoline-1-oxyl-3-oxide (TMA-PTIO), at different pH values, at which NO+ is, in aqueous solution, either highly unstable (pH 7.4) or relatively stable (pH 2.0). *NO donation by PAPA-NONOate, as indicated by TMA-PTIO decolorization, was similar at either pH and 3-acetamidomethyl-6-methoxycinnolinone (AMMC) was formed as the major product from AMK, at pH 7.4 more efficiently than at pH 2.0. At pH 2.0, TMA-PTIO decolorization by NO+SbF6- was much weaker than by PAPA-NONOate, but AMMC was produced at substantial rates, whereas neither TMA-PTIO decolorization nor AMMC formation was observed with the NO+ donor at pH 7.4. As NO+ is also stable in organic, especially aprotic solvents, NO+SbF6- was reacted with AMK in acetonitrile, ethanol, butanol, and ethyl acetate. In all these cases, AMMC was the only or major product. In ethyl acetate, N1-acetyl-5-methoxy-3-nitrokynuramine (AMNK) was also formed, presumably as a consequence of organic peroxides emerging in that solvent. Presence of tert-butylhydroperoxide in an ethanolic solution of NO+SbF6- and AMK also resulted in AMNK formation, in addition to AMMC and two red-fluoresecent, to date unknown products. However, hydrogen peroxide enhanced *NO-dependent AMMC production from AMK and also from N1-acetyl-N2-formyl-5-methoxykynuramine. HNO donation by Angeli's salt (Na2N2O3) also caused AMMC formation from AMK at pH 7.4, with a somewhat lower efficiency than PAPA-NONOate, but no AMNK nor any other product was detected. Therefore, all three NO congeners are, in principle, capable of nitrosating AMK and forming AMMC, but in biological material the reaction with NO+ is strongly limited by the extremely short life-time of this redox form.

The effect of melatonin and structural analogues on the lipid peroxidation of triglycerides enriched in omega-3 polyunsaturated fatty acids.

The lipid peroxidation of triglycerides enriched in polyunsaturated fatty acids was investigated by photoemission techniques and the TBARS assay. Butylated hydroxytoluene, 5-OH-tryptophan and N-acetylserotonin inhibited light emission and TBARS formation in a concentration dependent manner. However, it was enhanced in the presence of melatonin and 5-methoxytryptamine and was dependent on its concentration. The total relative luminic units were found to be lower in those systems incubated in the presence of butylated hydroxytoluene, N-acetylserotonin or 5-OH-tryptophan; this decreased proportionally to the concentration of the compound tested. The order of inhibition was 5-OH-tryptophan>N-acetylserotonin>butylated hydroxytoluene with the following IC50 values: 0.65, 6.5 and 9.0 mM respectively. The free-radical scavenging activity of the indole derivatives was also analyzed by the DPPH method, and the results indicate that 5-OH-tryptophan, and N-acetylserotonin exhibited a dose-dependent free-radical scavenging ability at all of the tested concentrations. Thus, at 10 microM concentration a decrease of 84.71% and 73.50% of initial DPPH was observed, compared to 51.00% of BHT. Melatonin and 5-methoxytriptamine decreased the initial concentration of DPPH only 1.85% and 5.0%, respectively. The possible formation of N(1)-acetyl-N(2) formyl-5-methoxykynuramine (AFMK) during lipid peroxidation of triglycerides enriched in PUFAs with cumene hydroperoxide in the presence of melatonin was also analyzed.

UV filter decomposition. A study of reactions of 4-(2-aminophenyl)-4-oxocrotonic acid with amino acids and antioxidants present in the human lens.

Deamination of UV filters, such as kynurenine (KN), in the human lens results in protein modification. Thermal reactions of the product of kynurenine deamination, 4-(2-aminophenyl)-4-oxocrotonic acid (CKA), with amino acids (histidine, lysine, methionine, tryptophan, tyrosine, cysteine) and antioxidants (ascorbate, NADH, glutathione reduced) were studied. The rate constants of the reactions under physiological conditions were measured. The rate constants of CKA addition to cysteine k(Cys)=36+/-4M(-1)s(-1) and to glutathione k(GSH)=2.1+/-0.2M(-1)s(-1) are 4-5 orders of magnitude higher than the rate constants of CKA reactions with the other amino acids and antioxidants. The Arrhenius parameters for k(Cys) and k(GSH) were determined: A(GSH)=(1.8+/-0.7)x10(5)M(-1)s(-1), E(GSH)=29.2+/-5.6kJmol(-1), A(Cys)=(2.7+/-0.9)x10(8)M(-1)s(-1), E(Cys)=40.4+/-5.7kJmol(-1). The large difference in frequency factors for k(Cys) and k(GSH) is attributed to steric hindrance, peculiar to the bulky GSH molecule.

Variations in activity and inhibition with pH: the protonated amine is the substrate for monoamine oxidase, but uncharged inhibitors bind better.

It has been accepted that, as required mechanistically, the neutral form of the amine is the substrate for monoamine oxidase, despite the amine pK (a) of above 9.5. The pH dependence of the kinetic parameters for kynuramine oxidation by purified human MAO-A and for phenylethylamine oxidation by MAO-B in granulocytes at pH values from 5 to 10 was consistent with the protonated amine being used. Deprotonation of a group of pK (a) = 7.1 in MAO-B and pK (a) = 7.5 +/- 0.1 (n = 4) in MAO-A was important for efficient catalysis. The K(i) values for two oxazolidinone inhibitors of MAO-A gave opposite pH-dependence indicating that the uncharged form of each inhibitor bound better than the charged form. Decreased pH induced a blue shift in the spectral maximum of MAO-A indicative of a more hydrophobic environment around the flavin, and also influenced the redox properties of the flavin.

Reactions of the melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK) with the ABTS cation radical: identification of new oxidation products.

The melatonin metabolite N1-acetyl-5-methoxykynuramine (AMK; 1), which was previously shown to be a potent radical scavenger, was oxidized using the ABTS cation radical [ABTS = 2,2'-azino-bis-(3-ethylbenzthiazoline-6-sulfonic acid)]. Several new oxidation products were obtained, which were separated by repeated chromatography and characterized by spectroscopic methods such as mass spectrometry (ESI-MS and ESI-HRMS), 1H-NMR and 13C-NMR, HMBC, HSQC, H,H COSY correlations and IR spectroscopy. The main products were oligomers of 1 (3 dimers, 1 trimer and 2 tetramers). In all cases, the amino group N2 was involved in the reactions. Two of the dimers turned out to be cis (2a) and trans (2b) isomers containing an azo bond. In the other dimer (3a), the nitrogen atom N2 was attached to atom C5 of the second aromatic amine, with loss of the methoxy group. In the trimer (5), one N2 formed a bridge to C5 of unit B, as in the respective dimer, while this one of C had bridged to C6 of B. One of the tetramers (6) was composed of a trimer 5 attached to N2 of a fourth 1 molecule via an azo bond as in 2a/b. In the other tetramer (7), an additional C-C bond between rings B and C in 6 is assumed. Although oligomers of AMK may only attain low in vivo concentrations, the types of reactions observed shed light on the physiologically possible metabolism of AMK once reacted with a free radical. The displacement of a methoxy group, rarely seen in the oxidation of methoxylated biomolecules, underlines the reactivity of AMK (1). Preliminary data show that, in the presence of ABTS cation radicals, AMK (1) can interact with side chains of aromatic amino acids, a finding which may be crucial for understanding to date unidentified protein modification by a melatonin metabolite detected earlier in experiments with radioactively labeled melatonin.