Browsing by Author "Robalo, Maria Paula"
Now showing 1 - 10 of 41
Results Per Page
Sort Options
- 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.
- Bacterial laccases: some recent advances and applicationsPublication . Martins, Lígia O.; Melo, Eduardo P.; Sanchez-Amat, Antonio; Robalo, Maria PaulaLaccases belong to the large family of multi-copper oxidases (MCOs) that couple the one-electron oxidation of substrates with the four-electron reduction of molecular oxygen to water. Because of their high relative non-specific oxidation capacity particularly on phenols and aromatic amines as well as the lack of requirement for expensive organic cofactors, they have found application in a large number of biotechnological fields. The vast majority of studies and applications were performed using fungal laccases, but bacterial laccases show interesting properties such as optimal temperature above 50 °C, optimal pH at the neutral to alkaline range, thermal and chemical stability and increased salt tolerance. Additionally, bacterial systems benefit from a wide range of molecular biology tools that facilitates their engineering and achievement of high yields of protein production and set-up of cost-effective bioprocesses. In this review we will provide up-to-date information on the distribution and putative physiological role of bacterial laccases and highlight their distinctive structural and biochemical properties, discuss the key role of copper in the biochemical properties, discuss thermostability determinants and, finally, review biotechnological applications with a focus on catalytic mechanisms on phenolics and aromatic amines.
- Benzo[c]thiophene chromophores linked to cationic Fe and Ru derivatives for NLO materials: synthesis characterization and quadratic hyperpolarizabilitiesPublication . Silva, Tiago J. L.; Mendes, Paulo J.; Garcia, M. Helena; Robalo, Maria Paula; Ramalho, J. P. Prates; Carvalho, A. J. Palace; Buechert, Marina; Wittenburg, Christian; Heck, Juergen5-Monocyclopentadienyliron(II)/ruthenium(II) complexes of the general formula [M(5-C5H5)(PP)(L1)][PF6] {M = Fe, PP = dppe; M = Ru, PP = dppe or 2PPh3; L1 = 5-[3-(thiophen-2-yl)benzo[c]thiophenyl]thiophene-2-carbonitrile} have been synthesized and studied to evaluate their molecular quadratic hyperpolarizabilities. The compounds were fully characterized by NMR, FTIR and UV/Vis spectroscopy and their electrochemical behaviour studied by cyclic voltammetry. Quadratic hyperpolarizabilities () were determined by hyper-Rayleigh scattering measurements at a fundamental wavelength of 1500 nm. Density functional theory calculations were employed to rationalize the second-order non-linear optical properties of these complexes.
- 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.
- Biological activity and cellular uptake of [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] complexPublication . Morais, Tania S.; Santos, Filipa; Côrte-Real, Leonor; Marques, Fernanda; Robalo, Maria Paula; Paulo J. Amorim Madeira; Garcia, M. HelenaAnticancer activity of the new [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] (Me(2)bpy = 4,4'-dimethyl-2,2'-bipyridine) complex was evaluated in vitro against several human cancer cell lines, namely A2780, A2780CisR, HT29, MCF7, MDAMB231 and PC3. Remarkably, the IC50 values, placed in the nanomolar and sub-micromolar range, largely exceeded the activity of cisplatin. Binding to human serum albumin, either HSA (human serum albumin) or HSA(faf) (fatty acid-free human serum albumin) does not affect the complex activity. Fluorescence studies revealed that the present ruthenium complex strongly quench the intrinsic fluorescence of albumin. Cell death by the [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] complex was reduced in the presence of endocytosis modulators and at low temperature, suggesting an energy-dependent mechanism consistent with endocytosis. On the whole, the biological activity evaluated herein suggests that the complex could be a promising anticancer agent. (C) 2013 Elsevier Inc. All rights reserved.
- DNA interaction and cytotoxicity studies of new ruthenium(II) cyclopentadienyl derivative complexescontaining heteroaromatic ligandsPublication . Moreno, Virtudes; Font-Bardia, Merce; Calvet, Teresa; Julia Lorenzo; Aviles, Francesc Xavier; Garcia, M. Helena; Morais, Tânia S.; Valente, Andreia; Robalo, Maria PaulaFour ruthenium(II) complexes with the formula [Ru(eta(5)-C(5)H(5))(PP)L][CF(3)SO(3)], being (PP = two triphenylphosphine molecules), L = 1-benzylimidazole, 1; (PP = two triphenylphosphine molecules), L = 2,2'bipyridine, 2; (PP = two triphenylphosphine molecules), L = 4-Methylpyridine, 3; (PP = 1,2-bis(diphenylphosphine) ethane), L = 4-Methylpyridine, 4, were prepared, in view to evaluate their potentialities as antitumor agents. The compounds were completely characterized by NMR spectroscopy and their crystal and molecular structures were determined by X-ray diffraction. Electrochemical studies were carried out giving for all the compounds quasi-reversible processes. The images obtained by atomic force microscopy (AFM) suggest interaction with pBR322 plasmid DNA. Measurements of the viscosity of solutions of free DNA and DNA incubated with different concentrations of the compounds confirmed this interaction. The cytotoxicity of compounds 1234 was much higher than that of cisplatin against human leukemia cancer cells (HL-60 cells). IC(50) values for all the compounds are in the range of submicromolar amounts. Apoptotic death percentage was also studied resulting similar than that of cisplatin. (C) 2010 Elsevier Inc. All rights reserved.
- Eco-friendly synthesis of indo dyes mediated by a bacterial laccasePublication . Sousa, Ana Catarina; Piedade, M. Fatima M.; Martins, Ligia O.; Robalo, Maria PaulaSeveral aminoindamine and indoaniline dyes were obtained in good to excellent yields (64-98%) by oxidative cross-coupling between 1,4-phenylenediamine (1), 4-aminophenol (2) or 2,5-diaminotoluene (3) and several meta- and meta, para-substituted couplers (4a-j) using a green biocatalyst, the bacterial enzyme CotA-laccase from Bacillus subtilis, in water and under mild conditions of pH and temperature. Our results show that the enzymatic route described represents an efficient and sustainable alternative to the chemical synthesis of indo dyes, with a potentially high impact in the cosmetic and hair dye industries.
- 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.
- Green extraction of high added value substances from spent coffee grounds: preliminary resultsPublication . Georgieva, Sílvia S.; Coelho, Jose; Campos, Filipe C.; Robalo, Maria Paula; Stateva, Roumiana P.A supercritical fluid extraction (SFE) with pure CO2 and ethanol as a co-solvent (10 %) of spent coffee grounds was carried out at temperatures from 313 K to 333 K, pressures up to 40.0 MPa and the results obtained were compared with those of the conventional Soxhlet method using n-hexane as a solvent. The best extraction yield (12.0 %) was achieved by SFE with CO2+10 % ethanol, compared to 9.5 - 10.7 % by SFE with pure CO2 and 10.4 % with n-hexane. The antioxidant potential was evaluated by DPPH method and the best antioxidant activity was exhibited by the spent coffee oil obtained by SFE, CO2+10 % ethanol. The quantification and the identification of the oils composition were carried out by NMR. The main constituents of the extracted oils referred to the triacylglycerols (TAGs, 98.9 - 96.8 %). Minor amounts of 1,2 diacylglycerols (1,2 DAGs, 2.7 - 0.68 %), and the diterpenes cafestol (6.02 - 2.26 %), 16-O-methylcafestol (4.86 - 2.00 %) and kahweol (2.11 - 0.71 %), as well as caffeine (1.45 - 0.1 %), were also present in all the extracts.