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A sustainable synthesis of asymmetric phenazines and phenoxazinones mediated by CotA-Laccase

Publication . Sousa, Ana Catarina; Oliveira, Maria Conceição; Martins, Lígia O.; Robalo, Maria Paula

An 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.

2018-02-01Journal article

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Engineering a bacterial DyP-Type peroxidase for enhanced oxidation of lignin-related phenolics at alkaline pH

Publication . 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.

2017-05Journal article

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Synthesis of Substituted 4-Arylamine-1,2-naphthoquinones in One-Pot Reactions Using CotA-Laccase as Biocatalyst

Publication . Sousa, Ana Catarina; Santos, Iolanda; Piedade, M. Fatima M.; Martins, Lígia O.; Robalo, M. Paula

An efficient and environmentally benign biocatalytic strategy for the synthesis of substituted 4-arylamino-1,2-naphthoquinones was developed, through a cross-coupling reaction in which the 1,2-naphthoquinone nucleus, formed in the biocatalytic process mediated by CotA-laccase from Bacillus subtilis, is the key synthetic intermediate. Electrochemical data and kinetic parameters were determined revealing a significant higher specificity of CotA-laccase for 4-amino-3-hydroxynaphthalene-1-sulfonic acid (AHNSA). This ability of CotA-laccase to discriminate between oxidisable aromatic amines allows the set-up of one-pot reactions in the presence of the enzyme, between AHNSA and a set of appropriate aromatic amines under mild reaction conditions.

2020-08-19Journal article

Open access

Donor functionalized iron(II) N-Heterocyclic carbene complexes in transfer hydrogenation reactions

Publication . Lopes, Rita; Raya-Baron, Alvaro; Robalo, M. Paula; S. Vinagreiro, Carolina; Barroso, Sonia; Romão, Maria João; Fernandez, Ignacio; Pereira, Mariette M.; Royo

Two piano-stool iron(II) complexes bearing N-heterocyclic carbene ligands outfitted with acetamide- and amine-pendant arms [Cp*Fe(NHC(R))(CO)I] {Cp* = eta(5)-tetramethylcyclopentadienyl; R = CH2CONEt2(3), (CH2)(2)NEt2(4)}, have been prepared and fully characterized. Their catalytic activity in transfer hydrogenation (TH) of ketones usingiPrOH as a hydrogen source and catalytic amounts of base (LiOtBu) has been explored, along with that of previously reported [CpFe(NHC(R))(CO)I] {R =nBu (5), (CH2)(2)OH (6), Et (7), and (CH2)(3)OH (8)} complexes containing hydroxyl and nonfunctionalized alkyl arms. Complex3displayed the highest catalytic activity of the whole series3-8, reaching a TOF(50)value of 533 h(-1). NMR monitoring of the stoichiometric reaction of3with LiOtBu, allowed the identification of a new species3'containing a deprotonated amidate moiety, which has been fully characterized by(1)H,C-13, and(15)N NMR. Finally, a green protocol for the reduction of ketones through TH using glycerol as a hydrogen source, under microwave irradiation in the presence of catalytic amounts of3and base has been developed.

2021-01-08Journal article

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