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- A sustainable synthesis of asymmetric phenazines and phenoxazinones mediated by CotA-LaccasePublication . Sousa, Ana Catarina; Oliveira, Maria Conceição; Martins, Lígia O.; Robalo, Maria PaulaAn efficient and sustainable one-step procedure for the synthesis of new asymmetric phenazines and phenoxazinones from commercially available ortho-substituted diamines and ortho-substituted hydroxyamines is reported. In this study we have expanded the substrate scope of CotA-laccase-catalyzed aerobic oxidations through the use of aromatic amines presenting variable functional groups, including N-substitution, contributing to the rational synthesis of different heterocyclic scaffolds. The transformations proceed smoothly through a cascade of oxidative reactions to the benzoquinonediimine intermediates followed by nucleophilic addition, intramolecular cyclization and aromatization, all performed in mild conditions.
- Laccases: versatile biocatalysts for the synthesis of heterocyclic coresPublication . Sousa, Ana Catarina; Martins, Lígia O.; Robalo, M. PaulaLaccases are multicopper oxidases that have shown a great potential in various biotechnological and green chemistry processes mainly due to their high relative non-specific oxidation of phenols, arylamines and some inorganic metals, and their high redox potentials that can span from 500 to 800 mV vs. SHE. Other advantages of laccases include the use of readily available oxygen as a second substrate, the formation of water as a side-product and no requirement for cofactors. Importantly, addition of low-molecular-weight redox mediators that act as electron shuttles, promoting the oxidation of complex bulky substrates and/or of higher redox potential than the enzymes themselves, can further expand their substrate scope, in the so-called laccase-mediated systems (LMS). Laccase bioprocesses can be designed for efficiency at both acidic and basic conditions since it is known that fungal and bacterial laccases exhibit distinct optimal pH values for the similar phenolic and aromatic amines. This review covers studies on the synthesis of five- and six-membered ring heterocyclic cores, such as benzimidazoles, benzofurans, benzothiazoles, quinazoline and quinazolinone, phenazine, phenoxazine, phenoxazinone and phenothiazine derivatives. The enzymes used and the reaction protocols are briefly outlined, and the mechanistic pathways described.
- Towards the rational biosynthesis of substituted phenazines and phenoxazinones by laccasesPublication . Sousa, Ana Catarina; Conceição Oliveira, M.; Martins, Lígia O.; Robalo, Maria PaulaLaccases are multi-copper oxidases that oxidise a wide range of substrates including phenol and aniline derivatives, which could be further involved in coupling reactions leading to the formation of dimeric and trimeric structures. This paper describes the enzyme-mediated dimerisation of several ortho and meta, para-disubstituted aromatic amines into phenazine ("head-to-tail" dimers) and phenoxazinone chromophores. The redox properties of substituted aromatic amines were studied by cyclic voltammetry and the kinetic constants of CotA and Trametes versicolor laccases were measured for selected aromatic amines. The structure of novel enzymatically synthesised phenazine and phenoxazinone dyes using CotA laccase was assessed by NMR and MS. Overall our data show that this enzymatic green process is an efficient alternative to the classic chemical oxidation of aromatic amines and phenols, with an impact on the broad field of applications of these heterocyclic compounds.
- Methyl syringate: An efficient phenolic mediator for bacterial and fungal laccasesPublication . Rosado, Tânia; Bernardo, Pedro; Koci, Kamila; Coelho, Ana V.; Robalo, Maria Paula; Martins, Lígia O.The aim of the present work is to provide insight into the mechanism of laccase reactions using syringyl-type mediators. We studied the pH dependence and the kinetics of oxidation of syringyl-type phenolics using the low CotA and the high redox potential TvL laccases. Additionally, the efficiency of these compounds as redox mediators for the oxidation of non-phenolic lignin units was tested at different pH values and increasing mediator/non-phenolic ratios. Finally, the intermediates and products of reactions were identified by LC-MS and H-1 NMR. These approaches allow concluding on the (1) mechanism involved in the oxidation of phenolics by bacterial laccases, (2) importance of the chemical nature and properties of phenolic mediators, (3) apparent independence of the enzyme's properties on the yields of non-phenolics conversion, (4) competitive routes involved in the catalytic cycle of the laccase-mediator system with several new C-O coupling type structures being proposed.
- Biocatalytic synthesis of 1,2-naphtoquinones derivatives mediated by CotA-laccasePublication . Sousa, Ana Catarina; Santos, Iolanda; Martins, Lígia O.; Robalo, Maria PaulaCatalysis is one of the cornerstones of our present economy and society and the formation of value-added products is many times directly dependent on catalytic technologies. Nowadays, there is a growing need for development of green strategies involving clean organic reactions, which do not use harmful organic solvents and toxic reagents. Amongst the many options available for a synthetic organic chemist, biocatalysis has emerged as one approach with an excellent potential. Enzyme-catalyzed reactions offer a number of advantages compared to the traditional chemistry-catalyzed reactions and biocatalytic methods impart a “greener” character to the synthesis. Laccases (EC1.10.3.2, p-diphenol:dioxygen oxidoreductases) are multicopper oxidoreductive enzymes which have proven to be versatile and highly/efficient biocatalyst for the synthesis of different value-added chemicals and pharmaceuticals.1 A large number of 1,2-naphthoquinones derivatives have been reported to show antitumor activities by inhibit on of multiple enzymes.2 In addition to their anticancer properties, the naphthoquinone framework has significance in the development of new substances with promising biological activities in other diseases like neurodegenerative and viral diseases.3 The formation of naphthoquinone frameworks is quit-well documented and reported methods include various approaches using organic solvents and different chemical oxidants.4 In this context it is still a challenge to explore alternative and more sustainable synthetic routes for these compounds. As a part of our going research program for exploring the catalytic properties of CotA-laccase, a bacterial laccase isolated from the Bacillus subtilis, we describe in the present communication a practical and simple oxidative CotA-laccase mediated eco-friendly method to obtain 1,2-naphthoquinones derivatives using mild aqueous conditions and O2 as oxidant. All compounds were isolated in good yields and fully characterized by FTIR, NMR and ESI techniques.
- Synthesis of azobenzene dyes mediated by cotA laccasePublication . Sousa, Ana Catarina; Baptista, Sara R.; Martins, Lígia O.; Robalo, Maria PaulaAn 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-ethylbenzo-thiazoline-6-sulfonic acid)) enablethe 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-potreaction. Amechanistic proposal for the formation of the azoderivatives is presented. Our strategy offers an alternative approach for the direct synthesis of azobenzene dyes, avoiding the harsh conditions generally required formost of the traditional synthetic methods.
- An enzymatic route to a benzocarbazole framework using bacterial CotA laccasePublication . Sousa, Ana Catarina; Piedade, Maria de Fátima M. M.; Martins, Lígia O.; Robalo, Maria PaulaThe CotA laccase-catalysed oxidation of the meta, para-disubstituted arylamine 2,4-diaminophenyldiamine delivers, under mild reaction conditions, a benzocarbazole derivative (1) (74% yield), a key structural motif of a diverse range of applications. This work extends the scope of aromatic frameworks obtained using these enzymes and represents a new efficient and clean method to construct in one step C-C and C-N bonds.
- Bacterial enzymes and multi-enzymatic systems for cleaning-up dyes from the environmentPublication . Mendes, Sónia; Robalo, Maria Paula; Martins, Lígia O.Synthetic dyes are xenobiotic compounds that are being increasingly used in several industries, with special emphasis in the paper, textile and leather industries. Over 100,000 commercial dyes exist today and more than 7 × 105 tons of dyestuff is produced annually, of which 1–1.5 × 105 tons is released into the wastewaters (Rai et al in Crit Rev Environ Sci Tecnhol 35:219–238, 2005). Among these, azo dyes, characterized by the presence of one or more azo groups (–N=N–), and anthraquinonic dyes represent the largest and most versatile groups.
- Wasteful azo dyes as a source of biologically active building blocksPublication . Fernandes, Ana; Pinto, Bruna; Bonardo, Lorenzo; Royo, Beatriz; Robalo, M. Paula; Martins, Lígia O.In this work, an environment-friendly enzymatic strategy was developed for the valorisation of dye-containing wastewaters. We set up biocatalytic processes for the conversion of azo dyes representative of the main classes used in the textile industry into valuable aromatic compounds: aromatic amines, phenoxazinones, phenazines, and naphthoquinones. First, purified preparations of PpAzoR azoreductase efficiently reduced mordant, acid, reactive, and direct azo dyes into aromatic amines, and CotA-laccase oxidised these compounds into phenazines, phenoxazinones, and naphthoquinones. Second, whole cells containing the overproduced enzymes were utilised in the two-step enzymatic conversion of the model mordant black 9 dye into sodium 2-amino-3-oxo-3H-phenoxazine-8-sulphonate, allowing to overcome the drawbacks associated with the use of expensive purified enzymes, co-factors, or exquisite reaction conditions. Third, cells immobilised in sodium alginate allowed recycling the biocatalysts and achieving very good to excellent final phenoxazine product yields (up to 80%) in water and with less impurities in the final reaction mixtures. Finally, one-pot systems using recycled immobilised cells co-producing both enzymes resulted in the highest phenoxazinone yields (90%) through the sequential use of static and stirring conditions, controlling the oxygenation of reaction mixtures and the successive activity of azoreductase (anaerobic) and laccase (aerobic).
- Engineering a bacterial DyP-Type peroxidase for enhanced oxidation of lignin-related phenolics at alkaline pHPublication . Brissos, Vânia; Tavares, Diogo; Sousa, Ana Catarina; Robalo, Maria Paula; Martins, Lígia O.Dye-decolorizing peroxidases (DyPs) are a family of microbial heme-containing peroxidases that show important properties for lignocellulose biorefineries due to their ability to oxidize lignin-related compounds. Directed evolution was used to improve the efficiency of the bacterial PpDyP from Pseudomonas putida MET94 for phenolic compounds. Three rounds of random mutagenesis by error prone PCR of the ppDyP gene followed by high-throughput screening allow identification of the 6E10 variant showing a 100-fold enhanced catalytic efficiency (k(ca)t/K-m) for 2,6-dimethoxyphenol (DMP), similar to that exhibited by fungal lignin peroxidases (similar to 10(5) M-1 s(-1)). The evolved variant showed additional improved efficiency for a number of syringyl-type phenolics, guaiacol, aromatic amines, Kraft lignin, and the lignin phenolic model dimer guaiacylglycerol-beta-guaiacyl ether. Importantly, variant 6E10 displayed optimal pH at 8.5, an upshift of 4 units in comparison to the wild type, showed resistance to hydrogen peroxide inactivation, and was produced at 2-fold higher yields. The acquired mutations in the course of the evolution affected three amino acid residues (E188K, A142V, and H125Y) situated at the surface of the enzyme, in the second shell of the heme cavity. Biochemical analysis of hit variants from the laboratory evolution, and single variants constructed using site-directed mutagenesis, unveiled the critical role of acquired mutations from the catalytic, stability, and structural viewpoints. We show that epistasis between A142V and E188K mutations is crucial to determine the substrate specificity of 6E10. Evidence suggests that ABTS and DMP oxidation occurs at the heme access channel. Details of the catalytic cycle of 6E10 were elucidated through transient kinetics, providing evidence for the formation of a reversible enzyme hydrogen peroxide complex (Compound 0) barely detected in the majority of heme peroxidases studied to date.