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.

Optimized synthesis of the melatonin metabolite N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK).

N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK) is the product of oxidative pyrrole ring cleavage of melatonin. AFMK and its deformylated derivative N(1)-acetyl-5-methoxykynuramine (AMK) are compounds for which there are increasing demands because of their antioxidant, immunomodulatory and anti-inflammatory properties. Here, we sought to determine the best reaction conditions for preparation of AFMK using chlorpromazine (CPZ) as a co-catalyst in the peroxidase-mediated oxidation of melatonin. The parameters studied were pH, identity and concentration of buffers, hydrogen peroxide (H(2)O(2)) and CPZ concentrations and the presence or absence of dissolved molecular oxygen in the reaction medium. The rate and efficiency of AFMK production were compared with a noncatalyzed method which uses a high concentration of H(2)O(2). We found that by using CPZ and bubbling molecular oxygen during the course of the reaction, the yield of AFMK was significantly increased (about 60%) and the reaction time decreased (about 30 min), as compared with the noncatalyzed reaction (yield 32% and reaction time 4 hr). Based on these data, we suggest that this could be a new, easily performed and efficient route for AFMK preparation. Additionally, we provide evidence that a radical chain reaction could be responsible for the formation of AFMK.

The effect of pH on horseradish peroxidase-catalyzed oxidation of melatonin: production of N1-acetyl-N2-5-methoxykynuramine versus radical-mediated degradation.

There is a growing body of evidence that melatonin and its oxidation product, N(1)-acetyl-N(2)-formyl-5-methoxykynuramine (AFMK), have anti-inflammatory properties. From a nutritional point of view, the discovery of melatonin in plant tissues emphasizes the importance of its relationship with plant peroxidases. Here we found that the pH of the reaction mixture has a profound influence in the reaction rate and products distribution when melatonin is oxidized by the plant enzyme horseradish peroxidase. At pH 5.5, 1 mm of melatonin was almost completely oxidized within 2 min, whereas only about 3% was consumed at pH 7.4. However, the relative yield of AFMK was higher in physiological pH. Radical-mediated oxidation products, including 2-hydroxymelatonin, a dimer of 2-hydroxymelatonin and O-demethylated dimer of melatonin account for the fast consumption of melatonin at pH 5.5. The higher production of AFMK at pH 7.4 was explained by the involvement of compound III of peroxidases as evidenced by spectral studies. On the other hand, the fast oxidative degradation at pH 5.5 was explained by the classic peroxidase cycle.

Kynurenamines as neural nitric oxide synthase inhibitors.

To find new compounds with potential neuroprotective activity, we have designed, synthesized, and characterized a series of neural nitric oxide synthase (nNOS) inhibitors with a kynurenamine structure. Among them, N-[3-(2-amino-5-methoxyphenyl)-3-oxopropyl]acetamide is the main melatonin metabolite in the brain and shows the highest activity in the series, with an inhibition percentage of 65% at a 1 mM concentration. The structure-activity relationship of the new series partially reflects that of the previously reported 2-acylamido-4-(2-amino-5-methoxyphenyl)-4-oxobutyric acids, endowed with a kynurenine-like structure. Structural comparisons between these new kinurenamine derivatives, kynurenines, and 1-acyl-3-(2-amino-5-methoxyphenyl)-4,5-dihydro-1H-pyrazole derivatives also reported confirm our previous model for the nNOS inhibition.

Synthesis of internal labeled standards of melatonin and its metabolite N1-acetyl-N2-formyl-5-methoxykynuramine for their quantification using an on-line liquid chromatography-electrospray tandem mass spectrometry system.

Melatonin (N-acetyl-5-methoxytryptamine) is implicated in physiologic changes related to light-dark cycles and has been recently found to display antioxidant properties. It is known that the reaction of melatonin with certain reactive oxygen and nitrogen species, such as hydrogen peroxide and singlet oxygen, produces N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK). We report herein on the development of a new liquid chromatography/tandem mass spectrometry (LC/ESI/MS-MS) assay to quantitatively determine melatonin and AFMK. The stable isotopic internal standard of melatonin-D3 was synthesized by the reaction of 5-methoxytryptamine with deuterated acetyl chloride (CD3COCl). Labeled AFMK (AFMK-D3) was obtained after photooxidation of melatonin-D3. The predominant ion [M + H]+ in the full scan mass spectra of melatonin, melatonin-D3, AFMK and AFMK-D3 were located, respectively, at m/z = 233, 236, 265 and 268. The collision-induced dissociation of the molecules revealed a predominant fragment at m/z = 174 for melatonin and melatonin-D3 (loss of the N-acetyl group), and at m/z = 178 for AFMK and AFMK-D3 (loss of both the N-acetyl and the N-formyl groups). The m/z transitions from 233 to 174 (melatonin), from 236 to 174 (melatonin-D3), from 265 to 178 (AFMK), and from 268 to 178 (AFMK-D3) were therefore chosen for the multiple reaction monitoring detection experiments, ensuring a high specificity and an accurate quantification of melatonin and AFMK in human plasma.

Oxidation of melatonin by singlet molecular oxygen (O2(1deltag)) produces N1-acetyl-N2-formyl-5-methoxykynurenine.

It has been shown that melatonin exhibits antioxidant properties. Chemical structures of some of the products formed by the interaction of melatonin with reactive oxygen and nitrogen species have been elucidated. Despite some evidence that the reaction of melatonin with singlet molecular oxygen (O2(1deltag)) produces N1-acetyl-N2-formyl-5-methoxykynurenine (AFMK), it has not been fully documented. In this investigation, melatonin was oxidized by photosensitization with methylene blue or by a clean chemical source of O2(1deltag), the thermodecomposition of N,N'-di(2,3-dihydroxypropyl)-1,4-naphtalenedipropanamide (DHPNO2). The resulting product was characterized by high performance liquid chromatography, coupled to electrospray ionization mass spectrometry and also by 1H, 13C and dept135 nuclear magnetic resonance spectroscopy. An isotopically labeled DHPN18O2 was also prepared and used as a chemical source of labeled 18[O2(1deltag)] to unequivocally characterize the end product. The results uncovered by this work confirm the hypothesis that oxidation of melatonin by O2(1deltag) produces AFMK.

Synthesis of 4,5-dimethoxykynuramine and its in vivo conversion to 6,7-dimethoxy-4-quinolinol.

4,5-Dimethoxykynuramine was synthesized in a three-step sequence originating with veratrole. Indirect evidence indicates that the drug was converted in vivo to the hypotensive agent 6,7-dimethoxy-4-quinolinol by the action of monoamine oxidase.