On the mechanism of the laccase-mediator system in the oxidation of lignin

In an effort to elucidate the role of phenolic and non-phenolic lignin subunits in a laccase mediator (LM) system, vanillyl alcohol was oxidized with laccase in the presence and absence of the mediator 1-hydroxybenzotriazol (HBT). Furthermore, the role of phenolic, aliphatic hydroxyl, and carboxylic acid moieties in lignin degradation was elucidated by selectively blocking them. The modified samples were then subjected to laccase and laccase-HBT treatments. On the basis of this data it was possible to establish the role of this mediator. HBT mediates the oxidation of lignin by inducing side-chain oxidation and oxygen-addition products rather than oxidative coupling reactions.     

Laccase-catalysed reaction between Meldrum's acid and catechols/hydroquinones – An investigation

Enzymatic oxidation of catechols/hydroquinones in the presence of Meldrum's acid as a nucleophile in aqueous solution has been investigated in detail by applying laccase as a catalyst. Optimization of the reaction through the use of HBT/ABTS/HPI as a mediator allowed a yield of more than 65% of the isolated product. We derived some new compounds with catechol/hydroquinone ring with moderate yields based on enzymatic synthesis in environmentally benign aqueous solution. These types of reactions represent a milestone along the path to future sustainable green chemistry.     

Biodegradation of Bisphenol A and Decolorization of Synthetic Dyes by Laccase from White-Rot Fungus, Trametes polyzona

Purified laccase from Trametes polyzona WR710-1 was used as biocatalyst for bisphenol A biodegradation and decolorization of synthetic dyes. Degradation of bisphenol A by laccase with or without redox mediator, 1-hydroxybenzotriazole (HBT) was studied. The quantitative analysis by HPLC showed that bisphenol A rapidly oxidized by laccase with HBT. Bisphenol A was completely removed within 3 h and 4-isopropenylphenol was found as the oxidative degradation product from bisphenol A when identified by GC-MS. All synthetic dyes used in this experiment, Bromophenol Blue, Remazol Brilliant Blue R, Methyl Orange, Relative Black 5, Congo Red, and Acridine Orange were decolorized by Trametes laccase and the percentage of decolorization increased when 2 mM HBT was added in the reaction mixture. This is the first report showing that laccase from T. polyzona is an affective enzyme having high potential for environmental detoxification, bisphenol A degradation and synthetic dye decolorization.     

The quest for new mild and selective modifications of natural structures: laccase-catalysed oxidation of ergot alkaloids leads to unexpected stereoselective C-4 hydroxylation

Laccase-catalysed oxidation of ergot alkaloids in the absence of chemical mediators allowed the unexpected isolation of the mono-hydroxylated derivatives of compounds 2-7. Structure determination by NMR techniques clearly indicated that hydroxylation took place at the C-4 benzylic position. Quite notably, the proposed protocol allowed, for the first time, functionalisation at the C-4 position of the ergoline skeleton. Depending on the absence or on the presence of a C-10 α-methoxy substituent, hydroxylation was either stereoselective (furnishing C-4α OH derivatives) or gave rise to a C-4α/C-4β OH mixture in a 2:1 ratio, respectively.     

Laccase-catalyzed conversion of green tea catechins in the presence of gallic acid to epitheaflagallin and epitheaflagallin 3-O-gallate

Green tea extract containing catechin mixtures was treated with gallic acid using laccase (EC 1.10.3.2) to improve its function as a food material. After enzymatic oxidation, two new products were detected on an HPLC chromatogram. These products were purified by extraction with ethyl acetate, sequential column chromatographies, and crystallization. On the basis of ¹H/¹³C NMR, MALDI-TOF/high resolution MS, and UV-visible absorption spectral analyses, they were confirmed to be epitheaflagallin (5) and epitheaflagallin 3-O-gallate (6). This enzymatic process allows us to preferentially convert catechin derivatives in a crude mixture of green tea into different compounds.     

Gas chromatography/mass spectrometric study of non-commercial C-4-substituted 1,4-dihydropyridines and their oxidized derivatives

A gas chromatography/mass spectrometry (GC/MS) method for the qualitative and quantitative determination of the calcium-channel antagonists C-4-substituted 1,4-dihydropyridines, and their corresponding N-ethyl derivatives, is presented. Also, the electrochemical oxidation and the reactivity of the compounds with alkyl radicals derived from 2,2'-azobis-(2-amidinopropane) were monitored by GC/MS. Mass spectral fragmentation patterns for the C-4-substituted 1,4-dihydropy-ridine parent drugs were significantly different from those of their oxidation products, generated either by electrochemical oxidation or by reaction with alkyl radicals. However, for N-ethyl-1,4-dihydropyridine compounds it was not possible to detect the final products (pyridinium salts) using these experimental conditions.     

Promoting laccase activity towards non-phenolic substrates: a mechanistic investigation with some laccase-mediator systems

The oxidation of benzyl alcohols with the enzyme laccase, under mediation by appropriate mediator compounds, yields carbonylic products, whereas laccase can not oxidise these non-phenolic substrates directly. The oxidation step is performed by the oxidised form of the mediator (Med(ox)), generated on its interaction with laccase. The Med(ox) can follow either an electron transfer (ET) or a radical hydrogen atom transfer (HAT) route of oxidation of the substrates. Experimental evidence is reported that enables unambiguous assessment of the occurrence of either one the oxidation routes with each of the investigated mediators, namely, ABTS, HBT, HPI and VLA. Support to the conclusions is provided by (i) investigating the intermolecular selectivity of oxidation with appropriate substrates, (ii) attempting Hammett correlations for the oxidation of a series of 4-X-substituted benzyl alcohols, (iii) measuring the kinetic isotope effect, (iv) investigating the product pattern with suitable probe precursors. Based on these points, a HAT mechanism results to be followed by the laccase-HBT, laccase-HPI and laccase-VLA systems, whereas an ET route appears feasible in the case of the laccase-ABTS system.     

Introducing carboxyl and aldehyde groups to softwood-derived cellulosic fibers by laccase/TEMPO-catalyzed oxidation

For more cost-effective and/or value-added utilization of cellulosic fibers in pulp and paper industry, fiber engineering is an important concept. Essentially, fibers can be engineered via various mechanical, chemical, and biological processes. In the current study, the combined use of laccase and TEMPO was applied to introduce carboxyl and aldehyde groups to softwood-derived cellulosic fibers (bleached softwood kraft pulp). The process conditions in preparation of the engineered fibers were optimized. Under the conditions studied, the maximum increases in carboxyl and aldehyde contents were 360 % and 225 %, respectively. The effectiveness of the laccase/TEMPO system could be well explained by the reaction cycles in catalytic oxidation pathways. The findings of the current work may provide useful insights into the enzymatic modification of cellulosic fibers for papermaking applications.     

Degradation of Polycyclic Aromatic Hydrocarbons by <Emphasis Type="Italic">Rigidoporus lignosus</Emphasis> and its Laccase in the Presence of Redox Mediators

The metabolism of polycyclic aromatic hydrocarbons (PAHs) was studied in vivo and in vitro in systems consisting of Rigidoporus lignosus and its laccase, in the presence of so-called “mediator” compounds. The static culture of the native fungal strain was able to metabolize anthracene and 2-methylanthracene, but not 9-nitroanthracene. The addition of redox mediators 2,2’-azino-bis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), 1-hydroxybenzotriazole (HBT) or violuric acid (VA) led to a significant increase in the degradation of substrates. The oxidation of PAHs was not significant when purified laccase was used without the addition of mediators. The addition of these compounds increased the oxidation of all substrates by approximately 70–80% after 72 h of incubation. The degradation rate was highest for 2-methylanthracene in the presence of VA.     

Excited State Intramolecular Proton Transfer in Ethynyl‐Extended Regioisomers of 2‐(2′‐Hydroxyphenyl)benzothiazole: Effects of the Position and Electronic Nature of Substituent Groups

Although the organic dyes based on excited state intramolecular proton transfer (ESIPT) mechanism have attracted significant attention, the structure‐property relationship of ESIPT dyes needs to be further exploited. In this paper, three series of ethynyl‐extended regioisomers of 2‐(2′‐hydroxyphenyl)benzothiazole (HBT), at the 3′‐, 4′‐ and 6‐positions, respectively, have been synthesized. Changes in the absorption and emission spectra were correlated with the position and electronic nature of the substituent groups. Although 4′‐ and 6‐substituted HBT derivatives exhibited absorption bands at longer wavelengths, the keto‐emission of 3′‐substituted HBT derivatives was found at a substantially longer wavelength. The gradual red‐shifted fluorescence emission was found for 3′‐substituted HBT derivatives where the electron‐donating nature of substituent group increased, which was opposite to what was observed for 4′‐ and 6‐substituted HBT derivatives. The results derived from the theoretical calculations were in conformity with the experimental observations. Our study could potentially provide experimental and theoretical basis for designing novel ESIPT dyes that possess unique fluorescent properties.     

Synthesis of 4＂-benzyloxyimino-4＂-deoxyavermectin B 1 a derivatives

Six new 4＂-benzyloxyimino-4＂-deoxyavermectin B la derivatives were synthesized from avermectin Bla by the selective protection of C-5-hydroxy group, oxidation of C-4＂-hydroxy group, and deprotection followed by reaction with O-substituted hydroxylamine hydrochlorides. Their structures were confirmed by IR, 1H NMR, 13C NMR and MS. Insecticidal activities of the derivatives against Phopalosiphum pseudobrassicae, Spodoptera exigua and Pluteua xylosteua were evaluated.     

Novel laccase redox mediators: spectral, electrochemical, and kinetic properties

The screening of potential redox mediators for laccase was performed using homogeneous enzyme preparations from Coriolus hirsutus and Coriolus zonatus. It was discovered that derivatives of 1-phenyl-3-methyl-pyrazolones were efficient substrates for the laccases. The characterization of two representatives of the 1-phenyl-pyrazolone class, sodium 1-phenyl-3-methyl-4- methylamino-pyrazolone-5-N(4)-methanesulfonate and 1-(3'-sulfophenyl)-3- methylpyrazolone-5, in the reaction catalyzed by laccase was carried out using spectral, electrochemical, and enzyme kinetics methods. The kinetic parameters for the oxidation of the newly discovered substrates were comparable with those for 2,2'-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation by laccase. Electrochemical experiments demonstrated that oxidation of these compounds yielded two high-potential intermediates capable of oxidizing veratryl alcohol, which was used as a lignin model substrate, to the corresponding aldehyde and acid. 1-(3'-Sulfophenyl)-3- methylpyrazolone-5 was about 30-40% as effective in degrading veratryl alcohol compared to ABTS as judged from high-performance liquid chromatography kinetic studies. 1-Phenyl-3-methyl-pyrazolones may be of commercial interest for oxidoreductase-catalyzed biodegradation of organic compounds.     

Measurement of nanomolar diphenols by substrate recycling coupled to a pH-sensitive electrode

A new enzyme amplification scheme based on the measurement of the pH-change generated in the laccase/glucose dehydrogenase substrate recycling reaction has been established. Quinone species formed after oxidation of diphenols by laccase are reduced by glucose dehydrogenase in the presence of glucose. Catalytic oxidation of glucose to gluconic acid via gluconolactone leads to a pH-decrease. The utility of the very simple pH-sensitive antimony electrode for monitoring the bienzyme reaction has been shown. The detection limit for norepinephrine and p-aminophenol measured by the bienzyme-modified antimony electrode was 1 nmol/L in the presence of glucose. The enhancement in sensitivity and analytical applications of the proposed scheme are discussed. Permanent address: Department of Biosensors, Research Center for Molecular Diagnostics and Therapy, Simpheropolsky Blvd. 8, 113149 Moscow, Russia     

Synthesis of Azobenzene Dyes Mediated by CotA Laccase

An eco‐friendly protocol for the synthesis of azobenzene dyes by oxidative coupling of primary aromatic amines is reported. As efficient biocatalytic systems, CotA laccase and CotA laccase/ABTS (2,2′‐azinobis(3‐ethylbenzothiazoline‐6‐sulfonic acid)) enable the oxidation of various substituted anilines, in aqueous medium, ambient atmosphere and under mild reaction conditions of pH and temperature. A series of azobenzene dyes were prepared in good to excellent yields in an one‐pot reaction. A mechanistic proposal for the formation of the azo derivatives is presented. Our strategy offers an alternative approach for the direct synthesis of azobenzene dyes, avoiding the harsh conditions generally required for most of the traditional synthetic methods.     

Laccase‐catalyzed Synthesis and Antioxidant Property of Poly(catechin)

Catechin was oxidatively polymerized by laccase in a mixture of a polar organic solvent and buffer to give a new class of flavonoid polymers. Myceliophthora laccase showed high catalytic activity for the polymerization. Using a mixed solvent of acetone and pH 5 acetate buffer efficiently produced the polymer. Under the selected conditions, DMF‐soluble polymers were obtained in good yields. Effects of reaction parameters on the yield, solubility, and molecular weight of the polymer have been systematically investigated. A radical species was detected in the polymer by ESR spectroscopy. The polymers showed greatly amplified superoxide scavenging activity and xanthine oxidase inhibitory activity compared with monomeric catechin.

Superoxide scavenging activity of poly(catechin)s, n = 3. ○: catechin, □: poly(catechin).     

Regioselective synthesis of 2,8-disubstituted 4-aminopyrido[3,2-d]pyrimidine-6-carboxylic acid methyl ester compounds

We report herein the synthesis of 4-amino-2,8-dichloropyrido[3,2-d]pyrimidine derivatives 2 and their regioselective diversification through S(N)Ar and metal-catalyzed cross-coupling reactions. While amination of 2 took place selectively at C-2, the regioselectivity of thiol or thiolate addition depended on the reaction conditions. Selective C-8 addition was obtained in DMF with Hünig's base and C-2 addition in (i)PrOH. These C-2 or C-8 regioselective thiolations provided an opportunistic way to selectively activate either of the two positions toward the metal-catalyzed cross-coupling reaction. The chloride could be efficiently substituted by Suzuki-Miyaura reaction and the sulfanyl group by Liebeskind-Srogl cross-coupling reaction, demonstrating the orthogonality of both reactive centers. The development of regioselective conditions for these different transformations yielded the synthesis of 4-amino-2,6,8-trisubstituted pyrido[3,2-d]pyrimidine derivatives, with various substituents.     

Synthetic modifications of ascomycin - I. A chemoselective removal of the cyclohexyl residue of ascomycin

An efficient semisynthetic preparation of des-28-(cyclohexyl)methylene-28-oxo-ascomycin derivatives starting from 24,33-O-bis(tert-butyldimethylsilyl)-ascomycin (1) is described. The strategy for preparing 28-oxo-ascomycin derivatives involves the reduction of C-22 carbonyl group, followed by 5-endo-cyclization of the resulting C-22 alcohol with the C-19/C-20 double bond using an oxymercuration reaction; ozonolysis of the C-28/C-29 double bond and regeneration of the C-19/C-20 double bond. Further, the 20-mercury-substituted ascomycin derivatives could be reduced to the corresponding metal free cyclic ethers using n-Bu₃SnH.     

Novel laccase redox mediators

The screening of potential redox mediators for laccase was performed using homogeneous enzyme preparations from Coriolus hirsutus and Coriolus zonatus. It was discovered that derivatives of 1-phenyl-3-methyl-pyrazolones were efficient substrates for the laccases. The characterization of two representatives of the 1-phenyl-pyrazolone class, sodium 1-phenyl-3-methyl-4-methylamino-pyrazolone-5-N(4)-methanesulfonate and 1-(3′-sulfophenyl)-3-methylpyrazolone-5, in the reaction catalyzed by laccase was carried out using spectral, electrochemical, and enzyme kinetics methods. The kinetic parameters for the oxidation of the newly discovered substrates were comparable with those for 2,2′-azino-bis (3-ethylbenzthiazoline-6-sulfonate) (ABTS) oxidation by laccase. Electrochemical experiments demonstrated that oxidation of these compounds yielded two high-potential intermediates capable of oxidizing veratryl alcohol, which was used as a lignin model substrate, to the corresponding aldehyde and acid. 1-(3′-Sulfophenyl)-3-methylpyrazolone-5 was about 30–40% as effective in degrading veratryl alcohol compared to ABTS as judged from high-performance liquid chromatography kinetic studies. 1-Phenyl-3-methyl-pyrazolones may be of commerical interest for oxidoreductase-catalyzed biodegradation of organic compounds.     

Spectrophotometric, EPR and kinetic characterisation of the >N-O* radical from 1-hydroxybenzotriazole, a key reactive species in mediated enzymatic oxidations

Characterisation of the aminoxyl (>N-O*) radical BTNO, generated from 1-hydroxybenzotriazole (HBT) by the one-electron oxidant CAN (a Ce(IV) salt), confirms BTNO as the reactive intermediate in oxidations run with the laccase/HBT system.