Christopher J. Cooksey

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.

Two novel stilbene-2-carboxylic acid phytoalexins from Cajanus cajan

Abstract Three phytoalexins were isolated from leaves of pigeon pea which had been challenged with Botrytis cinerea . One was identified as pinostrobin chalc

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.

In vitro assessment of the structure-activity relationship of tyrosinase-dependent cytotoxicity of a series of substituted phenols.

The rate of oxidation by purified mushroom tyrosinase of 30 compounds was measured by oximetry, and the tyrosinase-dependent cytotoxicity of each estimated in an in vitro assay using exposure of non-melanogenic cells to the agents in the presence and absence of tyrosinase. Cytotoxicity was estimated by immediate inhibition of DNA synthesis; 4-hydroxyanisole was used as the reference material. Compounds that were not oxidized by tyrosinase were found to be non-toxic but there was no direct relationship between the rate of oxidation and the relative cytotoxicity of those materials that acted as substrates for the enzyme. Thioethers were found to be more cytotoxic than the corresponding phenoxyethers. This was partly due to their greater rate of oxidation by tyrosinase and, in the case of propylthiophenol, the consequence of higher effective toxicity of the lipophilic species. The optimum chain length for the side chain of the oxyethers was three saturated carbon atoms and the toxicity appeared to be influenced by the lipophilicity of the compounds, possibly reflecting the relative lipid solubility of the putative toxic ortho-quinones generated from them. The maximum tyrosinase-dependent toxicity observed was in the range 5–6 times the relative toxicity of 4-hydroxyanisole. Sulphinyl and sulphonyl derivatives were inactive. In addition to oxyethers and thioethers, esters and glycosides of oxyethers were also examined and were found to be toxic in the presence of tyrosinase when hydrolysed. The succinates were found to be oxidized and toxic in our test system, suggesting that they rapidly underwent spontaneous hydrolysis. Oximetry data suggest that slight spontaneous hydrolysis of the other compounds occurs but they were not toxic in our assay. Ring-methylated phenoxyethers were oxidized relatively slowly and were non-toxic. Fluorine-substituted phenoxyethers were oxidized slightly more rapidly and exhibited clear toxicity in our system. Sesamol was oxidized to a black pigment but was non-toxic in our assay. A water-soluble vitamin E derivative was not oxidized and was non-toxic. Allylhydroquinone was not oxidized but exhibited significant direct toxicity.

Two isoprenylated isoflavone phytoalexins from Cajanus cajan

Abstract Four phytoalexins were isolated from sliced seeds of pigeonpea which had been incubated with its native microflora. Two were identified as the known pigeonpea phytoalexins, cajanin and cajanol, and the other two were characterized as new isoprenylated isoflavones.

A dienyl stilbene phytoalexin from arachis hypogaea

Abstract The novel stilbene, 3-isopentadienyl-4,3′,5′-trihydroxystilbene, was identified as the major antifungal component elicited by slicing imbibed kernels of 11 genotypes of groundnut. Incubation for 24 hr after slicing yielded values which varied between 38.5 and 105.8 μ of the compound/g of unimbibed kernels according to cultivar. After incubation for 48 hr, yields rose to between 89.5 and 189.7 μ/g. The compound was inhibitory to both spore germination and hyphal extension of the fungus, Aspergillus flavus, at 14.0 and 11.3 μ/ml, respectively.

Homolytic displacement at carbon : V. Formation of cyclopropylcarbinylsulphones and trichloroethylcyclopropanes from but-3-enyl cobaloximes by a novel process involving homolytic attack at the δ-carbon of the butenyl ligand

Abstract But-3-enyl- and substituted but-3-enylcobaloximes react with bromotrichlormethane (or trichloromethanesulphonyl chloride) and with 4-toluenesulphonyl chloride thermally or photochemically to give good yields of β,β,β-trichloroethylcyclopropanes and cyclopropylcarbinyl(tolyl)sulphones, respectively. The reactions proceed by a chain mechanism in which a key step is a novel process in which homolytic attack of a trichloromethyl or 4-toluenesulphonyl radical at the δ-carbon of the butenyl ligand leads to synchronous or subsequent attack of the incipient γ-carbon radical on the α-carbon, causing cyclisation and displacement of cobaloxime(II). The other propagation step involves the reaction of the cobaloxime(II) with the bromotrichloromethane, trichloromethanesulphonyl chloride or 4-toluenesulphonyl chloride to give the reactive organic radical.

Reaction kinetics of 4-methoxy ortho benzoquinone in relation to its mechanism of cytotoxicity: a pulse radiolysis study

Rate constants quantifying the reactivity of 4-methoxy ortho benzoquinone, formed in the metabolic activation of 4-hydroxyanisole, a possible melanocytotoxic drug under current assessment as a treatment for malignant melanoma, have been determined by pulse radiolysis. The quinone is reactive towards the thiols cysteine (k = 3.5x10(5)M-1sec-1), glutathione (k = 3.1x10(5)M-1sec-1) and dithiothreitol (k = 3.5x10(5)M-1sec-1), but relatively unreactive towards other nucleophiles such as arginine (k less than or equal to 1M-1sec-1) and glutamine (k less than or equal to 1M-1sec-1). Redox exchange with ascorbate also occurs (k = 1.0x10(4)M-1sec-1). In view of the low reactivity of 4-methoxy ortho benzosemiquinone towards oxygen (k less than or equal to 10(5)M-1sec-1) and the model lipid trans-2-butenoic acid (k less than or equal to 2x10(5)M-1sec-1), it is unlikely that initiation of lipid peroxidation by the semiquinone is a major source of cytotoxicity. A more likely toxicity pathway appears to be covalent addition reactions of 4-methoxy ortho benzoquinone with cellular nucleophiles, especially thiols, and/or redox exchange reactions of the quinone leading to antioxidant depletion.

On the Interaction of Anisyl-3,4-Semiquinone with Oxygen

Pulse radiolysis studies of anisyl-3,4-semiquinone, formed in the metabolic activation of 4-hydroxyanisole, a possible melanocytotoxic drug under current assessment as a treatment for malignant melanoma, have shown this semiquinone to be unreactive towards oxygen (k less than or equal to 10(5) M-1 s-1), although the reverse reaction of O2- with anisyl-3,4-quinone is very rapid (k = 8.7 x 10(8) M-1 s-1). Since 1,4 benzoquinone is also unreactive towards anisyl-3,4-semiquinone (k less than or equal to 10(5) M-1 s-1), the one-electron reduction potential, E17 (anisyl-3,4-quinone/anisyl-3,4-semiquinone), is likely to be considerably more positive than 0.1 V. This suggests that the cytotoxicity mechanism does not involve the generation of O2- and possible subsequent production of H2O2 and/or OH., leading to lipid peroxidation, as previously proposed, but rather involves as yet unknown reactions of anisyl-3,4-quinone. This quinone is unstable in water and its absorption spectrum was measured immediately (less than 0.1 s) following disproportionation of anisyl-3,4-semiquinone, before significant decay of the quinone had occurred.

Sucrose: A Constitutive Elicitor of Phytoalexin Synthesis

Extracts of seeds and leaves of the tropical legume Cajanus cajan (L.) Millsp. (the pigeon pea) elicited the accumulation of three phytoalexins when applied as droplets to superficially wounded leaves of the plant. The active component was purified and identified as sucrose. Phytoalexin accumulation was proportional to the logarithm of the concentration of sucrose applied, with maxima ranging from 338 to 455 micrograms per gram (fresh weight) of leaf tissue. The sucrose concentrations required to elicit half these amounts ranged from 20 to 35 micrograms per milliliter, but other sugars had little effect even at 1000 micrograms per milliliter. The elicitor activity of sucrose was abolished by actinomycin D, puromycin, and cycloheximide at a concentration of 10 micrograms per milliliter or greater, suggesting that gene derepression is required for expression of the phytoalexin response.