Christopher A. Ramsden

Evidence of the Indirect Formation of the Catecholic Intermediate Substrate Responsible for the Autoactivation Kinetics of Tyrosinase

Tyrosinase (EC 1.14.18.1) exhibits unusual kinetic properties in the oxidation of monohydric phenol substrates consisting of a lag period that increases with increasing substrate concentration. The cause of this is an autocatalytic process dependent on the generation of a dihydric phenol substrate, which acts as an activator of the enzyme. Experiments with N-substituted dihydric phenol substrates (N-methyldopamine,N-acetyldopamine) demonstrate that oxygen consumption is retarded in the N-acetyl substituted material due to a diminished rate of cyclization. The oxygen uptake exhibited a similar pattern when N-acetyltyramine was oxidized, and this was reflected by a prolongation of the lag period.N,N-Dipropyldopamine was oxidized with normal kinetics but with an oxygen stoichiometry of 0.5 mol of oxygen/mol of substrate. We show that this is the result of the formation of a stable indoliumolate product with oxidation-reduction properties that prevent the formation of dopaminochrome, thus blocking further stages in the tyrosinase-catalyzed oxidation. Evidence that the indoliumolate product is formed by cyclization of theortho-quinone is presented by pulse radiolysis studies, which demonstrate the formation of the ortho-quinone (by disproportionation of the corresponding semiquinones), which cyclizes to give the indoliumolate. The rate constant for cyclization was shown to be 48 s−1 (at pH 6.0). Tyrosinase-catalyzed oxidation of the monohydric phenol analogue,N,N-dimethyltyramine, was shown to require the addition of a dihydric phenol. Oxygen utilization then exhibited a stoichiometry of 1.0, indicating that the reactions proceed only as far as the cyclization. The analogous stable cyclic indoliumolate product was shown to be formed, with UV absorption and NMR spectra closely similar to the indoliumolate derived fromN,N-dipropyldopamine. This material was methylated by catechol O-methyltransferase but was unreactive to redox reagents. The formation of the cyclic product accounts for the indefinite lag when N,N-dimethyltyramine is used as the substrate for tyrosinase in the absence of a dihydric phenol cofactor.

Heterocyclic mesomeric betaines

Article de synthese traitant du classement de ces composes et de leurs reactions de cycloaddition

Quinone Chemistry and Melanogenesis

Publisher Summary The divergence of the melanogenic pathway occurs after the initial oxidation step that yields dopaquinone. The current analytical approach to the classification of melanins depends on the assessment of the comparative levels of degradation products that are considered characteristic of indoles and benzothiazine residues. Several physical methods for the analysis of melanins are available, including solid-state nuclear magnetic resonance (NMR) spectroscopy using 13 C and 15 N as probes, and electron paramagnetic resonance (EPR) spectroscopy. The most reliable method of quantitative analysis of eumelanins and pheomelanins relies on chemical degradation and high-performance liquid chromatography (HPLC) separation of the degradation products. The basis of this method is the yield of (1) pyrrole-2, 3, 5-tricarboxylic acid (PTCA) from 5, 6-dihydroxyindole-2-carboxylic acid (DHICA) (2.8%) and 5, 6-dihydroxyindole (DHI) (0.03%) after permanganate oxidation as an indication of eumelanin and (2) aminohydroxyphenylalanine (AHP) from benzothiazine derivatives by hydriodic acid hydrolysis as an indicator of pheomelanin content. These products are separated by HPLC and quantified by ultraviolet (UV) detection. Three important alternative modes of reaction of ortho-quinones in biological systems can be identified: (1) addition, (2) reduction, and (3) tautomerism. These pathways are illustrated in the chapter with selected examples.

Imidazolines and Pancreatic Hormone Secretiona

ABSTRACT: A range of imidazoline derivatives are known to be effective stimulators of insulin secretion, and this response correlates with closure of ATP‐sensitive potassium channels in the pancreatic β‐cell. However, mounting evidence indicates that potassium channel blockade may form only part of the mechanism by which imidazolines exert their effects on insulin secretion. Additionally, it remains unclear whether members of this class of drugs can bind directly to potassium channel components and whether occupation of a single binding site accounts for their functional activity. This review considers recent developments in the field and highlights evidence that does not fit readily with the concept that a single mechanism of action is sufficient to mediate the effects of imidazolines on pancreatic hormone secretion.

Melanogenesis-targeted anti-melanoma pro-drug development: effect of side-chain variations on the cytotoxicity of tyrosinase-generated ortho-quinones in a model screening system.

A set of 26 substituted phenols, 10 of which were synthesised in our laboratories, were tested for their rate of oxidation by mushroom tyrosinase in vitro as determined by oximetry and spectrophotometry and for their cytotoxic action in a model system. With one exception (4-hydroxybenzoic acid) all the agents tested were oxidised to the corresponding ortho-quinones. The maximum rates of oxidation varied between 15.1 +/- 0.59 nmoles oxygen consumed per minute (4-(2-thioethylthio)phenol) and 372.9 +/- 5.61 nmoles O2/ min. (4-(2-Hydroxyethylthio)phenol) in a reaction system comprising 300 units tyrosinase and 200 microM substrate. The rates of generation of quinone were in close agreement with these oximetric data. Some anomalies in oxygen stoichiometry were observed due to reoxidation of reaction products. Four categories of compounds were tested: those known to undergo side-chain cyclisation (such as tyrosine) (Group A), alkylphenols of increasing chain length with or without terminal hydroxyl groups (Group B), compounds with charged or bulky side-chains (Group C) and agents with oxy-, thio- and selenyl-ether side-chains (Groups D, E and F). In the majority of cases, the cytotoxicity, measured by the reduction of thymidine incorporation in cells exposed for 30 min to the agent in the presence of tyrosinase, reflected the rate of oxidation and is ascribed to the toxic action of the derived ortho-quinone. Tyrosinase-dependent cytotoxicity was absent in cyclising (Group A) and in Group C compounds. Toxicity, expressed by comparison with 4-hydroxyanisole (4HA) (IC50 = 11.7 microM), ranged between 0.36 (4-hydroxybenzyl alcohol) and 1.07 (3-(4-hydroxyphenyl)propanol) for Group B compounds, and be-tween 0.83 (4-ethoxyphenol) and 2.08 (4-(2-hydroxyethylthio)phenol) for groups D, E and F. Addition of glutathione to the toxicity assay system abrogated the cytotoxic action and, on the basis of spectrophotometric data, this is ascribed to the prevention of cellular thiol depletion by the ortho-quinone products of tyrosinase oxidation of the phenolic substrates. The lack of toxicity of the group C compounds may be due to the inability of their derived quinones to gain access to the cells. Addition of catalase or deferoxamine to the incubation medium was without effect on tyrosinase-dependent toxicity.

OXIDATIVE REARRANGEMENT OF IMINES TO FORMAMIDES USING SODIUM PERBORATE

Abstract Oxidation of C-aryl or alkyl-N-arylaldimines 6 or 20 by sodium perborate tetrahydrate (SPB) in trifluoroacetic acid solution gives rearranged N,N-disubstituted formamides 9 and 21. Yields are variable and solvent dependant with the best yields (50–60%) being obtained for electronically neutral C-aryl substituents or C-s-alkyl substituents. Product formation is rationalised in terms of an intermediate oxaziridine 5 that rearranges via acid catalysed ON bond cleavage. An alternative CO bond cleavage of these intermediates accounts for the formation of aldehydes, which are common by-products. Rearrangement appears to be favoured by N-aryl substituents and by C-substituents that do not stabilise a developing positive charge on carbon. Further support for an oxaziridine intermediate 5 is provided by the observation that MCPBA oxidation of benzaldehyde phenylimine gives rearranged N,N-diphenylformamide. Under the conditions of the SPB oxidative rearrangements, oxaziridine formation may well occur by initial formation of trifluoroperacetic acid. Stereochemical aspects of this novel rearrangement of aldimines 1 → 2 have been investigated using trans- and cis-myrtanal 25 and 30. The observed epimerisation using the N-4-tolyl imine of trans-myrtanal 26 is believed to arise from equilibration of the precursor imine 26 with the tautomeric enamine 35b.

Rearrangement and cyclo-α-elimination of N-substituted amidines using (diacetoxyiodo)benzene

The products of reaction of N-substituted amidines with (diacetoxyiodo)benzene are determined by the nature of the amidine substituents and the reaction temperature: rearrangement of N2-phenylfuran-2-carboximidamide provides a convenient route to N-(2-furyl)acetamide, whereas N-phenyl C-alkyl formimidamides cyclise to give benzimidazoles in good yield.

Heterocyclic rearrangements. Part XIV. Attempts to activate ring-opening–ring-closure rearrangements with carbon as the central atom

Various 3-substituted quaternised pyridines and 4-substituted oxazoles have been prepared and studied, with a view to discovering substituents appropriate for reactions involving opening of the pyridine or oxazole ring with closure onto the substituent to form a new ring at the 2-(pyridine) or 5-(oxazole) position. None of these potential rearrangements succeeded; the reasons for this are discussed together with the evidence that this provides for the mechanism of previously reported rearrangements.

Pulse radiolysis studies of ortho-quinone chemistry relevant to melanogenesis.

The contributions of pulse radiolysis towards characterisation of unstable ortho-quinones relevant to melanogenesis are reviewed. The quinones discussed include dopaquinone, the precursor of both eumelanogenesis and phaeomelanogenesis, and 5-S-cysteinyldopaquinone, an early component of the phaeomelanogenic pathway. Redox exchange between dopaquinone and 5-S-cysteinyldopa is shown to be a determinant of the balance between eumelanogenesis and phaeomelanogenesis. Ortho-quinones resulting from the oxidation of tertiary N,N-dialkylcatecholamines cyclise to redox-inactive betaines which fail to autoactivate tyrosinase. This is consistent with the dopa detected during melanogenesis catalysed by tyrosinase being formed indirectly by a combination of dopaquinone intramolecular reductive addition to form leucodopachrome (cyclodopa), followed by redox exchange between remaining dopaquinone and leucodopachrome. Rapid tautomerism of the ortho-quinone of 4-cyanomethylcatechol to a redox-inactive quinomethane likewise inhibits tyrosinase autoactivation. The incorporation of trihydric phenol moieties in melanin is modelled by the reactions of several ortho-quinones with phloroglucinol, which itself is not directly oxidised by tyrosinase due to the meta-positioning of the hydroxyl groups. The importance of a susceptibility towards nucleophilic attack as well as a propensity to undergo redox-exchange, in the chemistry of melanogenic ortho-quinones, is emphasised.

Affinity isolation of imidazoline binding proteins from rat brain using 5-amino-efaroxan as a ligand

We have employed an amino derivative of the imidazoline ligand, efaroxan, to isolate imidazoline binding proteins from solubilised extracts of rat brain, by affinity chromatography. A number of proteins were specifically retained on the affinity column and one of these was immunoreactive with an antiserum raised against the ion conducting pore component of the ATP‐sensitive potassium channel. Patch clamp experiments confirmed that, like its parent compound, amino‐efaroxan blocks ATP‐sensitive potassium channels in human pancreatic β‐cells and can stimulate the insulin secretion from these cells. The results reveal that a member of the ion conducting pore component family is strongly associated with imidazoline binding proteins in brain and in the endocrine pancreas.