Oxidation Of 4-methylcatechol Research Articles

New Synthesis of 2-Alkoxy-5-methyl-1,4-benzoquinones by the Oxidation of 4-Methylcatechol with Copper(II) Chloride in Alcohols

Abstract 4-Methylcatechol was oxidized with copper(II) chloride in various alcohols under oxygen to give the corresponding 2-alkoxy-5-methyl-1,4-benzoquinones.

Nidamental gland catechol oxidase in the little skate ( Raja erinacea): latency caused by an endogenous low molecular weight factor

1. 1. The enzymatic oxidation of o-diphenol substrates such as 4-methylcatechol by nidamental gland extracts from Raja erinacea is typically preceded by a latent period of several minutes. When aliquots of heat-denatured nidamental gland extract (HDE) were included in catechol oxidase assay mixtures, the latent period was extended in proportion to the concentration of HDE. Dialysis and acid hydrolysis eliminated the ability of HDE to extend the latent period. 2. 2. When nidamental gland extracts were subjected to gel filtration over Sephadex G-25, the period of oxidation latency was reduced eight-fold while catechol oxidase activity was not affected. 3. 3. When substrate concentration was elevated, the specific activity increased while the latent period decreased. When measured at differing substrate concentrations, latency was found to be inversely proportional to the rate of enzymatic oxidation. 4. 4. HDE caused a latent period to precede spontaneous oxidation of 4-methylcatechol in the absence of enzyme. The rate of non-enzymatic oxidation in the presence of HDE increased with 4-methylcatechol concentration while the latent period decreased proportionally. 5. 5. Addition of reduced glutathione to assay mixtures, like HDE, increased latency with no apparent effect on catechol oxidase activity. Oxidized glutathione had no apparent effect on either latency or oxidase activity. Other reductants such as dithiothreitol and NADPH also extended latency without affecting enzyme activity. 6. 6. These results indicate that Raja erinacea nidamental gland extracts contain a low molecular weight factor which causes a temporary latent period to precede enzymatic oxidation of 4-methylcatechol. Exogenously added reductants have a similar effect, indicating that the latency factor might be an endogenous reductant. 7. 7. The observation that the latency factor causes a latent period to precede both enzymatic and non-enzymatic oxidation indicates that the factor interacts with the substrate or with the quinones produced by oxidation, but not necessarily with the catechol oxidase.

Metabolism of p -Cresol by the Fungus Aspergillus fumigatus

The fungus Aspergillus fumigatus ATCC 28282 was shown to grow on p-cresol as its sole source of carbon and energy. A pathway for metabolism of this compound was proposed. This has protocatechuate as the ring-fission substrate with cleavage and metabolism by an ortho-fission pathway. The protocatechuate was formed by two alternative routes, either by initial attack on the methyl group, which is oxidized to carboxyl, followed by ring-hydroxylation, or by ring-hydroxylation as the first step with subsequent oxidation of 4-methylcatechol to the acid. The pathway was elucidated from several pieces of evidence. A number of compounds, including 4-hydroxybenzyl alcohol, 4-hydroxybenzaldehyde, 4-hydroxybenzoic acid, protocatechuic acid, protocatechualdehyde, and 4-methylcatechol, appeared transiently in the medium during growth on p-cresol. These compounds were oxidized without lag by p-cresol-grown cells but not by succinate-grown cells. Enzyme activities for most of the proposed steps were demonstrated in cell extracts after growth on p-cresol, and the products of these activities were identified. None of the activities were found in succinate-grown cells.

Latent egg capsule catechol oxidase in the little skate (Raja erinacea)

Abstract 1. 1. Treatment of crude catechol oxidase from Raja erinacea nidamental gland with α-chymotrypsin reduced the latency period following o- diphenol addition and increased initial rates of substrate oxidation [Koob and Cox (1985) Bull. Mt Desert Is. Biol. Lab.25, 132–134; Koob and Cox (1988) Biol. Bull.175, 202–211]. 2. 2. Nidamental gland extracts prepared wth proteinase inhibitor cocktail or with phenylmethylsulfonyl fluoride alone displayed rates of 4-methylcatechol oxidation 17 and 13% those of control values, respectively. 3. 3. Initial lag period duration was increased more than 10-fold in these extracts. 4. 4. α-Chymotrypsin had little effect on lag period or oxidation rates of extracts containing proteinase inhibitors. 5. 5. Likewise, α-chymotrypsin did not induce catechol oxidase activity in dialyzed inhibitor extracts. 6. 6. These results indicate that a serine enzyme is required for catechol oxidase activation and that α-chymotrypsin does not act directly on the latent oxidase, but rather upon another factor(s) involved in activation. 7. 7. Activation of this latent catechol oxidase therefore appears to involve a multi-step cascade which could serve to regulate catechol oxidase activity precisely during skate egg capsule formation.

Characteristics of tyrosinase in AOT–isooctane reverse micelles

Isooctane-AOT-H(2)O is a suitable system for studying enzyme behavior in organic solvents. Tyrosinase was able to catalyze a well-known reaction in aqueous medium: oxidation of 4-methylcatechol to yield 4-methyl-o-benzoquinone. This reaction was studied using the preceding ternary system with adequate amounts of each component to make up reverse micelles. 4-Methyl-o-benzoquinone stability was demonstrated in isooctane even at alkaline pH values. Apparent K(m) and V(max) were similar to those in water, but substrate inhibition was more evident. The pH and temperature appear to be shifted toward high and low values, respectively. Characteristic parameters of reverse micelles, omega(0) (= H(2)O/AOT) and percentage of H(2)O (v/v), were investigated. The results obtained showed that the steady-state rate varies either with omega(0) or with percentage of H(2)O. The variation observed with omega(0) showed an optimal value while an increase in percentage of H(2)O can lead to decreased or increased activity depending on substrate concentration.

Cooxidation of styrene by horseradish peroxidase and phenols: a biochemical model for protein-mediated cooxidation.

Styrene is oxidized to styrene oxide and benzaldehyde when incubated with horseradish peroxidase, H2O2, and 4-methylphenol. Styrene oxide is not formed in the absence of any of these reaction components or of molecular oxygen. The coupling products 2-(4-methylphenoxy)-1-phenylethane, 2-(4-methylphenoxy)-1-phenylethan-1-ol, and 2-(4-methylphenoxy)-2-phenylethan-1-ol are not formed, but the ortho-linked dimer of 4-methylphenol is a major product. The epoxide oxygen is labeled in the presence of 18O2 but not H218O2. Styrene oxide formation is not inhibited by mannitol or superoxide dismutase. The stereochemistry of trans-[1-2H]styrene is partially scrambled in the epoxide product. EPR signals attributable to the 2,4-dihydroxy-5-methylphenoxy radical, a product of the oxidation of 4-methylcatechol, are observed if Zn2+ is added to stabilize the radical. This radical is only detected in the presence of styrene. The results imply that styrene is epoxidized by the hydroperoxy radical generated by addition of molecular oxygen to the 4-methylphenoxy radical. The epoxidation mimics the chemistry proposed to occur in the protein-mediated cooxidation of styrene by hemoglobin and myoglobin.

Chemical and enzymatic oxidation of 4-methylcatechol in the presence and absence of l-serine. Spectrophotometric determination of intermediates

A pathway for 4-methylcatechol oxidation by tyrosinase (monophenol, dihydroxy- l-phenylalanine:oxygen oxidoreductase, EC 1.14.18.1) in the presence of l-serine is proposed. Characterization of intermediates in this oxidative reaction and stoichiometry determination have both been performed. It has been possible to detect spectrophotometrically 4-methyl- o-benzoquinone as the first intermediate in this pathway after oxidizing 4-methylcatechol with epidermis tyrosinase or sodium periodate at pH 6.5. The steps for 4-methylcatechol transformation in the presence of l-serine could be: 4-methylcatechol → 4-methyl- o-benzoquinone → 5-methyl-4 N-serine-catechol → 5-methyl-4 N-serine- o-benzoquinone. Matrix analysis of the spectra obtained with a rapid-scan spectrophotometer verified that 4-methyl- o-benzoquinone was transformed into 5-methyl-4 N-serine- o-benzoquinone at a constant ratio, the stoichiometry for this conversion being the equation: 2-(4- methyl-o- benzoquinone + l-serine → 4-methylcatechol + 5-methyl -4N- serine-o- benzoquinone .

Copper complexes which catalyze the air oxidation of 4-methyl catechol

A study of the air oxidation of 4-methyl catechol catalyzed by simple copper-amine complexes was performed in aqueous solutions at pH = 6.5. Simple bi-, tri- and tetra-nitrogen ligands were complexed to Cu 2+ ions. Initial rate kinetics of their catalytic oxidation of 4-methyl catechol was followed optically. Proton NMR linebroadening and optical spectral studies were performed on the Cu 2+-amine-catechol solutions and these spectral data were correlated with the kinetic studies.

A rapid assay for catechol oxidase and laccase using 2-nitro-5-thiobenzoic acid

The reaction between 4-methyl-1,2-benzoquinone or p-benzoquinone with 2-nitro-5-thiobenzoic acid anion (TNB) consumes 1 mol of quinone per mol of thiol yielding almost colorless Michael-type adducts. Based on this reaction a convenient and sensitive assay for polyphenol oxidases was developed. The method depends on following spectrophotometrically (λ = 412 nm) the decrease in the absorbance of the yellow-colored TNB due to a coupled reaction with the quinones generated by enzymatic oxidation of 4-methylcatechol (catechol oxidase) or 1,4-dihydroxybenzene (laccase). Catechol oxidase activity was measured in enzyme preparations from potato, muchroom, apple, quince, and spinach; laccase was assayed in preparations from banana, spruce needles, and Rhus atomaria. For both enzymes the absorbance at 412 nm decreased linearly with time over the major part of the reaction. The relation between rate and enzyme concentration was linear for all catechol oxidases. With laccases, however, a linear dependence was observed only for low concentrations. A comparison of the TNB assay with other presently used assays (O 2 electrode, spectrophotometric, and chronometric methods) showed that the different tests give results for enzyme activity which are in good agreement with each other. Based on oxygen consumption measured by the O 2 electrode method and quinone production measured with the TNB test, it was calculated that at the very initial stage of the reaction the various enzymes consume approximately 0.5 ml of O 2 per mol of quinone formed.

Reaction mechanism of grape catechol oxidase—a kinetic study

The initial velocity of the oxidation of 4-methylcatechol by grape catechol oxidase was determined. The kinetic analysis indicates that first there is random binding of an oxygen and a 4-methylcatechol molecule to the enzyme. Then one product molecule is released prior to the binding of second 4-methylcatechol molecule which is followed by the release of a second product molecule. The true K m values were determined; they were found to be 0.5 mM for oxygen and 17 mM for 4-methylcatechol.

Effect of Phloroglucinol and Resorcinol on the Clingstone Peach Polyphenol Oxidase-catalyzed Oxidation of 4-Methylcatechol

Phloroglucinol and resorcinol are not substrates for clingstone peach (Prunus persica) polyphenol oxidase, but they react with 4-methyl-o-quinone, produced either enzymatically or nonenzymatically, to give an intense red or red-brown color with a maximal absorption at about 470 nanometers. Several colored products were isolated from an ethyl acetate extract of the reaction by two-dimensional thin layer chromatography. Based on thin layer chromatographic and spectral studies of the enzymatic and nonenzymatic reactions, polyphenol oxidase does not play a role in the reaction between 4-methyl-o-quinone and phloroglucinol, resorcinol, d-catechin, or orcinol. In such reactions, the function of polyphenol oxidase is the formation of 4-methyl-o-quinone which then reacts nonenzymatically with the above phenols. Activation energies of both enzymatic and nonenzymatic reactions were determined.

OXIDATION OF PHENOLS BY YEAST

A strain of yeast which utilizes phenols was isolated from activated sludge for sewage treatment with a medium containing phenol as a sole carbon source. The yeast can oxidize p-cresol after the cells had been incubated in a medium containing phenol. An oxidation product from p-cresol, which accumulated in the reaction mixture, was isolated and crystallized. Ultraviolet, infrared, and nuclear magnetic resonance spectra, and the result of elemental analysis of the oxidation product assigned its structure to be 5-formyl-2-hydroxy-4-methyl-2, 4-pentadienoic acid. An identical compound was obtained by oxidation of 4-methylcatechol with yeast cells. These results suggest that, in the phenol-adapted yeast, an enzyme sequence is formed, by which p-cresol is oxidized to 4-methylcatechol and its benzene ring is cleaved between carbons 1 and 6.

Biochemistry of melanin formation.

Biochemistry of melanin formation.