Alcohol benzylic oxidase

Wines stored in vats lined with epoxy resins can present unusually high levels of benzoic aldehyde (several mg/L). Benzylic alcohol is both a plasticizer and diluent of these resins. Its conversion into benzaldehyde can be due (Blaise 1986) to the action of an exocellular enzyme of Botrytis cinerea called Alcohol Benzylic Oxidase (ABO, E.C. 1.1.3.7.) responsible for this oxidation process (Blaise and Brun 1986). 

Aryl alcohol oxidase from the ligninolytic fungus Pleurotus eryngii had a strong preference for benzylic and allylic alcohols, showing activity on phenyl-substituted benzyl, cinnamyl, naphthyl and 2,4-hexadien-l-ol [103,104]. Another aryl alcohol oxidase, vanillyl alcohol oxidase (VAO) from the ascomycete Penicillium simplicissimum catalyzed the oxidation of vanillyl alcohol and the demethylation of 4-(methoxymethyl)phenol to vanillin and 4-hydro-xybenzaldehyde. In addition, VAO also catalyzed deamination of vanillyl amine to vanillin, and hydroxylation and dehydrogenation of 4-alkylphenols. For the oxidation of 4-alkylphenol, the ratio between the alcohol and alkene product depended on the length and bulkiness of the alkyl side-chain [105,106]. 4-Ethylphenol and 4-propylphenol, were mainly converted to (R)-l-(4 -hydroxyphenyl) alcohols, whereas medium-chain 4-alkylphenols such as 4-butylphenol were converted to l-(4 -hydroxyphenyl)alkenes. 

Hydroxylation of the benzylic methyl group of tolbutamide, the preferred site of oxidative attack by CYP2C9 (22), generates hydroxytolbutamide. Hydroxytolbutamide is rapidly oxidized by other enzymes, presumably aldehyde oxidase and/or alcohol dehydrogenase (ALD), to form the major isolated metabolite, the benzoic acid analog. 

Horse liver alcohol dehydrogenase, F93W mutant with 1224 also mutated to G,A,V,L. hydride transfer from benzyl alcohol to NAD Heterotetrameric sarcosine oxidase of Arthrobacter sp. 1-IN, proton transfer from adduct of FAD with sarcosine-(CH3) and sarcosine-(CD3)... 

These systems are also described as normal copper proteins due to their conventional ESR features. In the oxidized state, their color is light blue (almost undetectable) due to weak d-d transitions of the single Cu ion. The coordination sphere around Cu, which has either square planar or distorted tetrahedral geometry, contains four ligands with N and/or 0 donor atoms [ 12, 22]. Representative examples of proteins with this active site structure (see Fig. 1) and their respective catalytic function include galactose oxidase (1) (oxidation of primary alcohols) [23,24], phenylalanine hydroxylase (hydroxy-lation of aromatic substrates) [25,26], dopamine- 6-hydroxylase (C-Hbond activation of benzylic substrates) [27] and CuZn superoxide dismutase (disproportionation of 02 superoxide anion) [28,29]. 

Pyrethroids from Chrysanthemic Acid, The unsaturated side chains of the allethrolone alcohol moieties of the natural pyrethrins are readily epoxidized by microsomal oxidases and converted to diols, thus detoxifying the insecticides. Esterification of chrysanthemic acid (9), R = CH3, with substituted benzyl alcohols produces useful insecticides barthrin [70-43-9], 2-chloro-3,4-methylenedioxybenzyl ( )-of,/ra/w-chrysanthemate, and dimethrin [70-38-2], 2,4-dimethylbenzyl ( )-og/rinsecticidal activity but are of very low mammalian toxicity, ie, rat oralLDBOs >20,000 mg/kg. 

An aryl-alcohol oxidase produced optimally under carbon limitation from Bjerkandera adusta oxidized a number of benzyl alcohols including 4-methoxybenzyl alcohol, 3,4-dimethoxybenzyl alcohol (veratryl alcohol), and 4-hydroxy-3-methoxybenzyl alcohol, with the production of H202 from 02 monosaccharides were not oxidized (Muheim et al. 1990). An aryl-alcohol oxidase from Pleurotus eryngii is a flavoprotein with range of substrates comparable to that from B. adusta (Guillen et al. 1992). 

One molar aqueous ethylene glycol has been converted to glycolaldehyde in 90% yield using an alcohol oxidase, a method said to give better selectivity than chemical methods.193 Laccase has been used with oxygen in the presence of an azine to convert benzyl alcohols to aldehydes in 87-100% yields.194 (For more on biocatalysis, see Chap. 9.)... 

The direct transformation of alcohols to the corresponding amines is of growing interest because alcohols are easily available or accessible by chemical means. Amination of alcohols is usually catalyzed by transition metals at high temperatures and elevated pressures. Unfortunately, there is no enzyme known today that allows this particular functional group interconversion (FGl) in one step. Consequently, a multi-enzyme cascade was set up for the amination of alcohols as demonstrated for various benzylic and cinnamic alcohols under physiological conditions [24] aerobic alcohol oxidation toward the aldehyde was performed via a galactose oxidase originating from Fusarium (NRRL 2903 [25]) followed by an in situ co-TA-catalyzed reductive amination step (Scheme 4.5). 

A very similar process is used to catalyze the oxidation of aromatic and aliphatic alcohols to their corresponding aldehydes. An alcohol oxidase enzyme isolated from Pichia pastoris placed in a two-phase system will readily produce benzaldehyde from benzyl alcohol. While this oxidation is slow and inefficient in aqueous systems, oxidation in a two-phase system increased yield by a factor of nine and reaction time was shortened greatly [74]. 

Duff, S.J.B., W.D. Murray, Oxidation of benzyl alcohol by whole cells of Pichia pastoris and by alcohol oxidase in aqueous and nonaqueous reaction media, Biotech-nol. Bioeng., 34, p. 153, 1989. 

Two phenoxyl radical complexes [Cu (2 )N03] and [Zn (2 )N03] oxidize benzyl alcohol to benzaldehyde and have been studied as models for the enzyme galactose oxidase (GO). GO contains a dipeptide unit (3) in which a tyrosine residue is covalently bound to an adjacent cysteine residue and which is similar to (2), the tyrosyl radical in (3) also being bound to the Cu centre (see Figure 1). Second-order kinetics were observed with respect to [Zn°(2 )N03]+ and there was no evidence of redox reaction at the zinc site, suggesting that a dimeric form of the complex is active however, the reaction of [Cu H2 )N03]+ with benzyl alcohol is first order in the metal complex and [Cu (2H)]+ is identified as a product, suggesting a formal 2e /2H+ mechanism in which the monomeric form coordinates the alcohol in the manner believed to operate for G0. 2... 

Benzyl alcohol has also been employed as substrate with the methylotrophic yeast Pichia pastoris, in the presence of methanol, that induces the production of nonspecific alcohol oxidases able to convert benzyl alcohol to benzaldehyde. Very recently, in situ product removal approaches (adsorption and/or pervaporation) have been exploited with this system. In particular, adsorption on hydrophobic resin in a solid-liquid two-phase partitioning bioreactor afforded yields of up to 14g in a 5-1 reactor [104,105]. 

The experiments by Canonica et al. (1981) suggest a third plausible formation of simple isoquinoHnes with no carbon-1 substituent in plants mainly producing alkaloids of the benzylisoquinoline type. Treatment of the benzylisoquinoline N-methylcoclaurine with ascorbic acid oxidase, an enzyme widespread in plants, resulted in benzyhc fission giving about 40% yield of corypalline, its quaternary 1,2-dehydroderivative and benzyl alcohol. Thus, both isoquinolones and simple isoquinolines may be formed directly by the action of oxidative enzymes in benzyl-isoquinoline-producing plants. 

Scheme 5.13 Successful salen-based galactose oxidase biomimetic benzylic alcohol oxidation.

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