The catabolism of melatonin, whether naturally occurring or ingested, takes place via two pathways: ∼ 70 % can be accounted for by conjugation (sulpho-and glucurono-conjugation), and ∼ 30 % by oxidation. It is commonly thought that the interferoninduced enzyme indoleamine 2,3-dioxygenase (EC 1.13.11.42), which oxidizes tryptophan, is also responsible for the oxidation of 5-hydroxytryptamine (serotonin) and its derivative, melatonin. Using the recombinant enzyme expressed in Escherichia coli, we show in the present work that indoleamine 2,3-dioxygenase indeed cleaves tryptophan; however, under the same conditions, it is incapable of cleaving the two other indoleamines. By contrast, myeloperoxidase (EC 1.11.1.7) is capable of cleaving the indole moiety of melatonin. However, when using the peroxidase conditions of assay -with H 2 O 2 as co-substrate -indoleamine 2,3-dioxygenase is able to cleave melatonin into its main metabolite, a kynurenine derivative. The present work establishes that the oxidative metabolism of melatonin is due, in the presence of H 2 O 2 , to the activities of both myeloperoxidase and indoleamine 2,3-dioxygenase (with lower potency), since both enzymes have K m values for melatonin in the micromolar range. Under these conditions, several indolic compounds can be cleaved by both enzymes, such as tryptamine and 5-hydroxytryptamine. Furthermore, melatonin metabolism results in a kynurenine derivative, the pharmacological action of which remains to be studied, and could amplify the mechanisms of action of melatonin.
The standard method of peptide library synthesis involves coupling steps in which a single amino acid is reacted with a mixture of resin-bound amino acids. The more recently described positional scanning strategy (in which each position in the peptide sequence is occupied in turn by a single residue) is different since it involves the coupling of mixtures of amino acids to mixtures of resin-bound amino acids. In the present study, we analyze the compounds produced under these conditions measuring coupling rates and amounts of formed products, using mainly UV, HPLC, LC/MS and MS/MS techniques. Our data do not permit to conclude that the resulting libraries are complete. Indeed, our analytical data indicate that a large part of the di-, tri- and tetrapeptides synthesized with this method are not present in the final mixture. Although chemical compensation (in which poor coupling kinetics is compensated by a larger excess of the incoming amino acid) has been thought to counterbalance these biases, our experiments show that the compensation method does not take into account the crucial influence of the resin-bound amino acid and that even the dipeptide libraries obtained in this way are far from completeness. The present work provides strong evidence that the coupling of mixtures of amino acids to resin-bound residues, which is required by the positional scanning strategy, results in incomplete and/or non-equimolar libraries. It also clearly confirms that coupling rates in solid-phase peptide synthesis are dependent on the nature of both the incoming and the immobilized amino acid.
Parallel synthesis techniques aim to prepare collections of single compounds which, once tested, can easily be identified by their sole location in the synthesic array. On the other hand, true combinatorial chemistry produces libraries of compounds as mixtures of variable size which require a deconvolution procedure for identification of the active hits or leads. In the latter case, analytical methods are crucial for the success of the strategy and mass spectrometry plays a major role. If the goal is to identify all the library components, including expected products as well as by-products, various mass spectrometric techniques may be necessary. Library components can be separated according to their mass by increasing mass resolution or by their elution time by coupling liquid chromatography and mass spectrometry. The efficiency of such separation techniques are discussed as a function of the size and the degeneracy of the library. Library members possess common structural features which impart similar fragmentation patterns after ionization in the gas phase. This feature can be exploited by tandem mass spectrometry to specifically detect subfamilies of products. Examples of precursor ion scans, product ion scans and constant neutral loss scans will be shown that facilitate partial characterization of libraries. To solve the difficult problem of the quantitative analysis of libraries, i.e., to evaluate their equimolarity, the use of an evaporative light scattering detector (ELSD) or a chemiluminescent nitrogen detector (CLND) is suggested as more appropriate.
Reaction of 5-hydroxy or 5-acetoxy flavones with NaB& in CF3COOH provides zwitterionic boroflavylium complexes instead of expected reduction products. lH, I3C, I9F, I l B N M R and mass spectrometry of these complexes are described.
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