Estudo da imobilização de proteases para a síntese de oligolisinas

Enzymatic synthesis of peptides using proteases has attracted a great deal of attention in recent years. One key challenge in peptide synthesis is to find supports for protease immobilization capable of working in aqueous medium at high performance, producing watersoluble oligopeptides. At present, few reports have been described using this strategy. Therefore, the aim of this thesis was to immobilize proteases applying different methods (Immobilization by covalent bound, entrapment onto polymeric gels of PVA and immobilization on glycidil metacrylate magnetic nanoparticles) in order to produce water-soluble oligopeptides derived from lysine. Three different proteases were used: trypsin, α-chymotrypsin and bromelain. According to immobilization strategies associated to the type of protease employed, trypsin-resin systems showed the best performance in terms of hydrolytic activity and oligopeptides synthesis. Hydrolytic activities of the free and immobilized enzymes were determined spectrophotometrically based on the absorbance change at 660 nm at 25 °C (Casein method). Calculations of oligolysine yield and average degree of polymerization (DPavg) were monitored by 1H-NMR analysis. Trypsin was covalently immobilized onto four different resins (Amberzyme, Eupergit C, Eupergit CM and Grace 192). Maximum yield of bound protein was 92 mg/g, 82 mg/g and 60 mg/g support for each resin respectively. The effectiveness of these systems (Trypsin-resins) was evaluated by hydrolysis of casein and synthesis of water-soluble oligolysine. Most systems were capable of catalyzing oligopeptide synthesis in aqueous medium, albeit at different efficiencies, namely: 40, 37 and 35% for Amberzyme, Eupergit C and Eupergit CM, respectively, in comparison with free enzyme. These systems produced oligomers in only 1 hour with DPavg higher than free enzyme. Among these systems, the Eupergit C-Trypsin system showed greater efficiency than others in terms of hydrolytic activity and thermal stability. However, this did not occur for oligolysine synthesis. Trypsin-Amberzyme proved to be more successful in oligopeptide synthesis, and exhibited excellent reusability, since it retained 90% of its initial hydrolytic and synthetic activity after 7 reuses. Trypsin hydrophobic interactions with Amberzyme support are responsible for protecting against strong enzyme conformational changes in the medium. In addition, the high concentration of oxirane groups on the surface promoted multi-covalent linking and, consequently, prevented the immobilized enzyme from leaching. The aforementioned results suggest that immobilized Trypsin on the supports evaluated can be efficiently used for oligopeptides synthesis in aqueous media

Micromorphology and spectroscopic characteristics of organic matter in waterlogged podzols of the upper Amazon basin

In waterlogged environments of the upper Amazon basin, organic matter is a major driver in the podzolisation of clay-depleted laterites, especially through its ability to weather clay minerals and chelate metals. Its structure in eight organic-rich samples collected at the margin and in the centre of the podzolic area of a soil sequence was investigated. The samples illustrate the main steps in the development of waterlogged podzols and belong either to eluviated topsoil A horizons or to illuviated subsoil Bhs, Bh and 2BCs horizons. Organic matter micromorphology was described, and the overall molecular structure of their clay size fractions was assessed using Fourier transform infrared (FTIR) spectroscopy and cross polarization/magic angle spinning (CP/MAS) C-13 nuclear magnetic resonance (NMR). Organic features of the horizons strongly vary both vertically and laterally in the sequence. Topsoil A horizons are dominated by organic residues juxtaposed to clean sands with a major aliphatic contribution. In the subsoil, numerous coatings, characteristic of illuviation processes, are observed in the following horizons: (i) At the margin and bottom parts of the podzolic area, dark brown organic compounds of low aromacity with abundant oxygen-containing groups accumulate in Bhs and 2BCs horizons. Their spectroscopic features agree with the observation of cracked coatings in 2BCs and the presence of organometallic complexes, whose abundance decreases towards low lying positions. (ii) By contrast, black organic compounds of high aromacity with few chelating functions accumulate as coatings and infills in the overlying sandy Bh horizon of well-expressed waterlogged podzols. (c) 2008 Elsevier B.V. All rights reserved.

Highly corrosion resistant siloxane-polymethyl methacrylate hybrid coatings

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Synthesis, spectral and thermal studies on dicarboxylate-bridged palladium(II) coordination polymers. Part I

This work describes the synthesis, IR and (13)C CPMAS NMR spectroscopic as well the thermal characterization of the new dicarboxylate complexes [Pd(2)(ox)(2)(4,4'-bipy)]n (1), [Pd(2)(ox)(2)(bpe)](n) (2) and [Pd(2)(ox)(2)(pz)](n) (3) {ox = oxalate, bipy = 4,4'-bipyridine, bpe = 1,2-bis(4-pyridyl)ethane, pz = pyrazine}. TG experiments reveal that compounds 1-3 undergo thermal decomposition in three steps. Metal palladium was the final product of the thermal decompositions, which was identified by X-ray powder diffraction.

Synthesis, spectral and thermal studies on dicarboxylate-bridged palladium(II) coordination polymers. Part II

The synthesis and thermal behavior of the new [Pd(fum)(bipy)] (n) center dot 2nH(2)O (1), [Pd(fum)(bpe)] (n) center dot nH(2)O (2) and [Pd(fum)(pz)] (n) center dot 3nH(2)O (3) {bipy = 4,4'-bipyridine, bpe = 1,2-bis(4-pyridyl)ethene and pz = pyrazine} fumarate complexes are described in this work as well their characterization by IR and (13)C CPMAS NMR spectroscopies. TG curves showed that the compounds released organic ligands and lattice water molecules in the temperature range of 46-491 A degrees C. In all the cases, metallic palladium was identified as the final residue.

Dichloro(cyclohexilidene-1-methylene)(phenyl)Te(IV). Looking for the theoretical treatment

C(13)H(16)Cl(2)Te,M(r)=370.76,P2(1)/a, a = 8.1833(8), b = 8.4163(8), c = 20.787(2) A, beta = 99.52(1)degrees, Z = 4, R(1) = 0,0275. The primary coordination around the Te(IV) atom is consistent with a pseudo-trigonal bipyramidal bond configuration with two Cl atoms occupying axial positions while the C atoms and the lone pair of electrons occupy the equatorial positions. The Te(IV) atom is involved in an intermolecular secondary interaction resulting in the self assembly of zigzag-chains supramolecular array. In order to determine the theoretical basis set for the Te atom which leads to the best agreement with the experimental data, a large series of geometry optimizations were performed on dichloro dimethyl Te(IV), as a model compound, and the results compared with the mean distances and angles obtained from 45 X-ray structures. The Ahlrichs basis set plus the Hay & Wadt ECP was selected and used for a series of calculations performed on the title compound.

Evolution of rheological properties and local structure during gelation of siloxane-polymethylmethacrylate hybrid materials

Hybrid siloxane-polymethylmethacrylate (PMMA) nanocomposites with covalent bonds between the inorganic (siloxane) and organic (polymer) phases were prepared by the sot gel process through hydrolysis and polycondensation of 3-(trimethoxysilyl)propylmethacrylate (TMSM) and polymerization of methylmethacrylate (MMA) using benzoyl peroxide (BPO) as initiator. The effect of MMA, BPO and water contents on the viscoelastic behaviour of these materials was analysed during gelation by dynamic rheological measurements. The changes in storage (G') and loss moduli (G), complex viscosity (eta*) and phase angle (6) were measured as a function of the reaction time showing the viscous character of the sot in the initial step of gelation and its progressive transformation to an elastic gel. This study was complemented by Si-29 and C-13 solid-state nuclear magnetic resonance (NMR/MAS) measurements of dried gel. The analysis of the experimental results shows that linear chains are formed in the initial step of the gelation followed by a growth of branched structures and formation of a three-dimensional network. Near the gel point this hybrid material demonstrates the typical scaling behaviour expected from percolation theory.

Anxiolytic effects of erythrinian alkaloids from Erythrina mulungu

One new erythrinian alkaloid derivative, (+)-11 alpha-hydroxyerythravine (1), and the known (+)-erythravine (2) and (+)-alpha-hydroxyerysotrine (3) were isolated from the flowers of Erythrina mulungu. Their structures were determined by spectroscopic/spectrometric data interpretation of H-1, C-13, and 2D NMR and MS experiments. The relative configuration was established by NOESY analysis, while the conformation adopted by these molecules was evaluated through molecular modeling studies and coupling constants obtained by NMR analysis. Furthermore, the anxiolytic effects of the E. mulungu aqueous alcoholic crude extract and of the purified alkaloids were evaluated using the elevated T-maze test.

Studies on the constituents of a Brazilian folk infusion. Isolation and structure elucidation of new triterpene saponins from Ilex amara leaves

The isolation of three new triterpene saponins 3beta-O-beta-D-glucopyranosyl-(1-->3)-alpha-L-2-O-acetylara-binopyranosylolean-12-en-28-oic acid 28-O-beta-D-glucopyranosyl ester (2), 3beta-O-beta-D-glucopyranosyl-(1-->2)-alpha-L-O-arabinopyranosylurs-12-en-28-oic acid (3), and 3beta-O-beta-D-glucopyranosyl-(1-->2)-beta-D-O-galactopyranosylurs-12-en-28-oic acid (4) together with five known saponins and one flavonoid glycoside from the aqueous infusion of flex amara (Vellozo) Loes. leaves is reported. All structures were elucidated by spectroscopic methods, including the concerted application of one-dimensional (H-1, TOCSY, C-13, and C-13 DEPT NMR) and two-dimensional NMR techniques (DQF-COSY, HSQC, and HMBC).

Solid-state and solution structural study of acetylacetone-modified tin(IV) chloride used as a precursor of SnO2 nanoparticles prepared by a sol-gel route

The effect of addition of different amounts of acetylacetone (acacH) on the species formed at room temperature and after thermohydrolysis at 70 degreesC for 30 and 120 min of ethanolic SnCl4.5H(2)O solutions is followed by EXAFS spectroscopy at the Sn K-edge. We show that thermohydrolyzed solutions are a mixture of SnO2 nanoparticles and soluble tin polynuclear species. The complexation of the tin molecular precursors by acetylacetonate ligands is evidenced by H-1, C-13, and Sn-119 NMR spectroscopy and EXAFS for a acacH/Sn ratio higher than 2. Single crystals are isolated from solution and the structure, determined by X-ray diffraction, is built up from monomeric Cl-3(H2O)Sn(acac)-H2O units bridged together by hydrogen bonding. The acacH/Sn ratio in solution controls the polycondensation of the hydrolyzed species but not the crystallite size of the SnO2 nanoparticles (similar to2 nm). Because of the major presence of chelated tin mono- and dimeric complexes in solution for acacH/Sn > 2, the condensation is almost inhibited, meanwhile the decrease of amount of chelated complexes for the acacH/Sn < 2 gives rise to an increase of the number of nanoparticles.

Structural features of phosphate and sulfate modified zirconia prepared by sol-gel route

This paper describes the effect of sulfate, phosphate and nitrate complexing ligands on the structural features of amorphous xerogels and on the crystallization of metastable zirconia phases during the xerogel-ceramic conversion. Powdered samples were prepared by a sol-gel route using zirconyl chloride precursors chemically modified by complexing ligands. The structural evolution of ZrO2 phases as function of firing temperature was analyzed by XRPD, EXAFS and P-13 NMR/MAS. The experimental results show the formation of metastable t-ZrO2 during the low firing temperature of xerogels modified by sulfate or phosphate groups. The martensitic tetragonal-monoclinic transformation occurs during desorption of sulfate groups. The largest temperature interval of stability of metastable tetragonal zirconia was observed for phosphate-modified xerogels.

A laser-induced fluorescence spectroscopic study of organic matter in a Brazilian Oxisol under different tillage systems

Laser-induced fluorescence (LIF) spectroscopy has been proposed as new method for determining the degree of humification of organic matter (OM) in whole soils. It can be also used to analyze the OM in whole soils containing large amounts of paramagnetic materials, and which are neither feasible to Electron Paramagnetic Resonance (EPR) nor to C-13 Nuclear Magnetic Resonance (NMR) spectroscopy. In the present study, 3 LIF spectroscopy was used to investigate the OM in a Brazilian Oxisol containing high concentration of Fe+3. Soil samples were collected from two areas under conventional tillage (CT), two areas under no-till management (NT) and from a non-cultivated (NC) area under natural vegetation. The results of LIF spectroscopic analysis of the top layer (0-5 cm) of whole soils showed a less aromatic OM in the non-cultivated than in the cultivated soils. This is consistent with data corresponding to HA samples extracted from the same soils and analyzed by EPR, NMR and conventional fluorescence spectroscopy. The OM of whole soils at 5-10 and 10-20 cm depth was also characterized by LIF spectroscopy.Analysis of samples of NT and NC soils showed a higher OM aromatic content at depth. This is a consequence of the accumulation of plant residues at the soil surface in quantities that are too large for microorganisms to metabolize fully, thus, resulting in less aromatic or less hurnified humic substances. In deeper soil layers, the input of residues was lower and further decomposition of humic substances by microorganisms continued, and the aromaticity and degree of humification increased with soil depth. This data indicates that the gradient of humification of OM in the NT soil was similar to those observed in natural soils. Nevertheless, the degree of humification of the OM in the soils under no-till management varied less than that corresponding to non-cultivated soils. This may be because the former have been managed under these practices for only 5 years, in contrast to the continuous humification process occurring in the natural soils. on the other band, LIF spectroscopic analysis of the CT soils showed less pronounced changes or no change in the degree of humification with depth. This indicates that the ploughing and harrowing involved in CT lead to homogenization of the soil and thereby also of the degree of humification of OM throughout the profile. (c) 2006 Elsevier B.V. All rights reserved.

Cleavage of the dimeric cyclopalladated [Pd(N,C-dmba)(mu-X)](2), (dmba = N,N-dimethylbenzylamine; X = SCN and NCO) by diphosphines. Palladium(II) compounds with distinct structures in the solid-state and in solution

The reactions of the pseudohalide-bridged dimer [Pd(N,C-dmba)(mu -SCN)](2) (1) (dmba = N,N-dimethylbenzylamine) with cis-Ph2PCH=CHPPh2 (cis-dppet) (1:1 molar ratio) and of [Pd(N,C-dmba)(mu -NCO)](2) (2) with Ph2PCH2CH2PPh2 (dppe) (1:2 molar ratio) gave mononuclear [Pd(C-dmba)(SCN)(cis-dppet)].H2O (1a) and [Pd(C-dmba)(NCO)(dppe)] (2a), respectively, with the diphosphines acting as chelating ligands. Reaction of (2) with Fe(C5H4PPh2)(2) (dppf) (1:1 molar ratio) yielded [{Pd(N,C-dmba)(NCO)}(2)(mu -dppf)] (2b), a bimetallic species containing two palladium atoms bridged by the diphosphine, whereas reaction in a 1:2 molar ratio gave the mononuclear [Pd(N,C-dmba)(dppf)][NCO]. CH2Cl2 (2c), with the diphosphine acting as a chelating ligand. The compounds have been characterized by elemental analysis, i.r., P-31{H-1}, C-13- and H-1-n.m.r. spectroscopies. Conductivity measurements together with spectroscopic data showed that (1a) and (2a) do not have the same structure in the solid state and in MeCl solution, whereas for compounds (2b) and (2c) no structural changes were observed when the solids were dissolved in MeCl.

Tetrahydroisoquinoline alkaloids and 2-deoxyribonolactones from Aristolochia arcuata

2-Deoxyribonolactones and four tetrahydroisoquinoline alkaloids were isolated from the acetone extract of the leaves of Aristolochia arcuata Mast., together with pinitol, sequoyitol, glycerol, fructose, sucrose, eupomatenoid-7, salsolinol, and 6,7-dihydroxyl,1-dimethyl-1,2,3,4-tetrahydroisoquinoline. Their structures were determined on the basis of spectroscopic methods, mainly using H-1, C-13, N-15, and P-31 NMR. (C) 2003 Elsevier B.V. Ltd. All rights reserved.

Eu3+ doped polyphosphate-aminosilane organic-inorganic hybrids

The preparation and characterization of new Eu3+ doped polyphosphate-aminosilane hybrids xerogels is reported. Eu3+ D-5(0) emission quantum efficiency ranges from 0.41 to 0.54 depending on the SUP ratio. These rather high values are due to the substitution of phosphate and amino groups for water in the Eu3+ coordination shell. Raman and Si-29 and C-13 CP-MAS NMR results suggest that no strong interaction exists between the polyphosphate and the siloxane parts. Not fully condensed siloxane colloidal domains seem to be homogeneously distributed in the polyphosphate network. Good optical quality and favorable Eu3+ spectroscopic characteristics suggest these new hybrids as good hosts for lanthanide ions in optical devices. (C) 2003 Published by Elsevier B.V.