HRP-catalyzed oxidation of phenols

The origin of Groups I and II is otherwise rationalized in terms of the Marcus formalism (54) as in the case of HRP-catalyzed oxidation of phenols (125,126). The data in Fig. 11 are plotted to highlight the inverted Marcus region in reaction (37). The following standard assumptions have been made in the calculations (53,54) ... 

The data in Pig. 11 were fitted to Eq. (42), and the best-fit values for ( o os) and the reorganization parameter k equal (1.9 0.4) x 10 s and 0.45 0.04 eV, respectively. The latter is close to that found in HRP-catalyzed oxidation of phenols (125). Group II ferrocenes underscore the inverted region which was not clearly observed in the case of HRP-catalyzed oxidation of phenols (125). 

CL reaction can be catalyzed by enzymes other than HRP (e.g., microperoxidase and catalase) and by other substances [hemoglobin, cytochrome c, Fe(III), and other metal complexes]. The presence of suitable molecules such as phenols (p-iodophenol), naphthols (l-bromo-2-naphthol), or amines (p-anisidine) increases the light production deriving from the HRP-catalyzed oxidation of luminol and produces glow-type kinetics [6, 7], The use of other enzymes, such as glucose-6-phosphate dehydrogenase [38-41], P-galactosidase [42], and xanthine oxidase [43-46], as CL labels has been reported. 

Scheme 4 HRP-catalyzed oxidation of 2,4,6-trimethyl phenol with in situ electrogeneration of hydrogen peroxide [157].

A bienzymatic system was developed as catalyst for the oxidative polymerization of phenols.18 The HRP-catalyzed polymerization of phenol in the presence of glucose oxidase and glucose gave the polymer in a moderate yield, in which hydrogen peroxide was formed in situ by the oxidative reaction of glucose catalyzed by glucose oxidase. 

The enzymatic reaction kinetics on the HRP-catalyzed oxidation of p-cresol in aqueous 1,4-dioxane or methanol showed that the cataljdic turnover niunber and Michaelis constant were larger than those in water (235). Numerical and Monte Carlo simulations of the peroxidase-catalyzed polymerization of phenols were demonstrated (236). The simulations predicted the monomer reactivity and polymer molecular weight, leading to synthesis of polymers with specific molecular weight and index. In an aqueous 1,4-dioxane, the formation of monomer aggregate was observed (237), which might elucidate the specific polymerization behaviors in such a medium. 

Oxidation of phenols and aromatic amines using HRP is generally of little synthetic value, as oligomers and polymers are the main products (5, 260). Under certain conditions oxidative coupling of phenols or naphthols to give biaryls can be achieved, but with low selectivity (262). In contrast, HRP can catalyze a number of useful oxidative N-and 0-deaIkyIation reactions that are relatively difficult to carry out synthetically. This area has been described in detail by Meunier (263). A method for the preparation of optically active hydroperoxides using HRP C has been developed (264). Optically pure (S)-hydroperoxides... 

The classic reaction catalyzed by peroxidases is the one-electron oxidation of phenols. The guaiacol assay for peroxidase activity, an assay that derives from the earliest observation of peroxidase activity [70], involves the one-electron oxidation of guaiacol to a free radical that undergoes subsequent radical-radical combination to give a colored dimeric product (Fig. 5.13) [71]. The ability of substituted phenols to act as substrates and reduce HRP Compound I is controlled by the electronic nature of the substituent [72], At pH 7.0, the linear Hammett correlation observed between the substituent a value and the rate of Compound I reduction adheres to the equation log kx/ku = 6.92a, where kx is the rate of the... 

HRP catalyzes the oxidative dehydrogenation of a wide range of electron-rich aromatic compounds. The result of this radical formation pathway is dimerization and subsequent oligomerization of the substrates [76-78]. Peroxidases have been used to catalyze polymerizations of phenols (e.g. p-cresol and guaiacol) and aromatic amines (e.g. aniline, and o-phenyldiamine) [79, 80]. N- and O-dealkylations are also useful electron transfer reactions catalyzed by peroxidases. These reactions are used in industrial wastewater treatment and may have synthetic applications [81]. 

Comparison of the rate constants of [(TMP)Fe =0] + and HRP compound I was further extended to reactions with a series of sulfides [108]. HRP is known to convert thioanisole to the corresponding sulfoxide [109], although peroxidases typically catalyze two sequential one-electron oxidations such as one-electron oxidation of phenol derivatives to phenoxy radicals [110], The yield of the sulfoxide from the stoichiometric reaction of HRP compound I with thioanisole is only 25 + 5 %. The sulfoxidation involves oxygen-transfer from an oxoferryl species to sulfide, because in H2 02 has been shown to be incorporated into the product sulfoxide [108, 111], The initial rapid conversion of compound I to compound II (fci) by thioanisole is followed by further reduction to the ferric resting state, as is found for reactions with DMA [108, 112, 113]. A linear correlation between log A i and E°ox for the reactions of HRP compound I with thioanisoles (Figure 2) is readily combined with the relationship for DMA (Figure la) into a single, common relationship (Eq. 8) [108]. 

Horseradish peroxidase (HRP, EC 1.11.1.7) catalyzes the ( -dependent oxidation of phenols and amines forming colored polymeric products via radical intermediates. This reaction has been used to detect phenol, bilirubin and aminopyrine (Renneberg et al., 1982). The hydrogen peroxide required was either injected into the measuring cell or generated in the enzyme membrane itself. For the latter reaction, GOD was coimmobilized with HRP. 

The enzymatic polymerization of phenols in aqueous solutions often resulted in low yield of the insoluble polymer. The peroxidase-catalyzed polymerization of phenol took place in presence of 2,6-di-O-methyl-a-cyclodextrin (DM-a-CD) in buffer [32], Only a catalytic amount of DM-a-CD was necessary to induce the polymerization efficiently. Even for water-insoluble m-substituted phenols, the addition of 2,6-di-0-methyl-(3-cyclodextrin (DM-P-CD) enabled the enzymatic polymerization in a buffer [33]. The water-soluble complex of the monomer and DM-P-CD was formed, which was polymerized by HRP to give a soluble polymer. Coniferyl alcohol was oxidatively polymerized in the presence of a-CD in an aqueous solution [34],... 

The mechanistic study on the regioselectivity of the HRP-catalyzed oxidative polymerization was performed by using in situ NMR spectroscopy [41, 42], In the polymerization of 8-hydroxyquinoline-5-sulfonate, the 2, 4, and 7-positions were involved in the oxidative coupling with the order of preference being 7 > 2 > 4. The polymerizability of phenols via HRP catalysis was evaluated by the initial reaction rate [43]. Phenols with electron-donating groups were consumed much... 

Enzyme-Catalyzed Polymerization. Horseradish peroxidase (HRP) has been used for the oxidation of a wide variety of compounds, eg, phenol derivatives in the presence of hydroperoxide (44). HRP is a kind of heme glycoprotein that catalyzes the oxidation of phenol to phenoxy radicals. Subsequently, the resulting phenoxy radicals couple each other to oligo or polyphenol derivatives step by step (see Oxidative Polymerization). 

Peroxidase-catalyzed grafting of polyphenols on lignin has been attempted by HRP-catalyzed polymerization of p-cresol with lignin in the aqueous 1,4-dioxane or reverse micellar system. (288-290). The monomer was incorporated into lignin by the oxidative coupling between the monomer and the phenolic moiety of lignin. 

The principle of the polymerization of phenols catalyzed by a peroxidase is explained in the following text for the enzyme horseradish peroxidase (HRP). The mechanism of the HRP catalysis is fairly well understood and has been the subject of many investigations [24,51-54]. HRP catalyzes the one-electron oxidation of phenols by a peroxide to form the corresponding phenoxy radicals. Usually, hydrogen peroxide is used as oxidizing reagent. During this process, two water molecules are formed [55] (Eq. 1) ... 

Fig. 3 HRP-catalyzed oxidative polymerization of a water-soluble phenol-cyclodextrin...

Functionahzed polymers can be prepared from m-substituted phenols the HRP-catalyzed oxidative polymerization of m-ethynylphenol (76) using hydrogen peroxide in a solution of methanol/phosphate buffer (pH 7, 50 50 vol %) under air resulted in a phenol polymer having the ethynyl groups unaffected in the side chain [132]. hi contrast, a reaction with a conventional oxidation catalyst (Cu(I)Cl/TEED) led to coupling of the acetylene moiety to produce bis(3-hydroxyphenyl)butadiyne (78, Scheme 17) [132]. 

The emission yield from the horseradish peroxidase (HRP)-catalyzed luminol oxidations can be kicreased as much as a thousandfold upon addition of substituted phenols, eg, -iodophenol, -phenylphenol, or 6-hydroxybenzothiazole (119). Enhanced chemiluminescence, as this phenomenon is termed, has been the basis for several very sensitive immunometric assays that surpass the sensitivity of radioassay (120) techniques and has also been developed for detection of nucleic acid probes ia dot-slot. Southern, and Northern blot formats (121). 

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