New oxomolybdenum(VI) hybrid materials

In this thesis, 2,2’-bipyridine (bipy), di-tert-butyl-2,2’-bipyridine (di-t-Bubipy), 2,2’-bipyridine-5,5’-dicarboxylic acid (H2bpdc), 2-[3(5)-pyrazolyl]pyridine (pzpy) and 2-(1-pentyl-3-pyrazolyl)pyridine (pent-pp) ligands were used as the N,N-chelate ligands in the formation of discrete [MoO2Cl2L]-type complexes. These complexes were employed as precursors for the preparation in aqueous media of oxomolybdenum(VI) products with a wide range of structural diversity. Three distinct heating methods were studied: hydrothermal, reflux or microwave-assisted synthesis. An alternative reaction with the inorganic molybdenum(VI) trioxide (MoO3) and the ligands di-t-Bu-bipy, H2bpdc and pzpy was also investigated under hydrothermal conditions. The distinct nature of the N,N-chelate ligands and/or the heating method employed promoted the isolation of a series of new oxomolybdenum(VI) hybrid materials that clearly reflected the strong structure-directing influence of these ligands. Thus, this thesis describes the synthesis and characterization of the discrete mononuclear [MoO2Cl2(pent-pp)], the dinuclear [Mo2O6(di-t-Bu-bipy)2] and the octanuclear [Mo8O22(OH)4(di-t-Bu-bipy)4] complexes as well as the highly unique polymeric materials {[MoO3(bipy)][MoO3(H2O)]}n, (DMA)[MoO3(Hbpdc)]·nH2O, [Mo3O9(pzpy)]n and [Mo2O6(pent-pp)]n (fine structural details of compound [Mo2O6(pent-pp)]n are presently unknown; however, characterization data strongly pointed toward a polymeric oxide hybrid compound). The catalytic behaviour of the discrete complexes and the polymeric compounds was tested in olefin epoxidation reactions. Compounds [Mo3O9(pzpy)]n and [Mo2O6(pent-pp)]n acted as sources of soluble active species that where identified as the oxodiperoxido complexes [MoO(O2)2(pzpy)] and [MoO(O2)2(pent-pp)], respectively. The majority of the compounds here presented were fully characterized by using solid-state techniques, namely elemental analyses, thermogravimetry, FT-IR, solid-state NMR, electron microscopy and powder X-ray diffraction (both from laboratory and/or synchrotron sources).

Structural analysis of glycans and development of microarrays from Helcobacter pylori cell surface glycome

Helicobacter pylori is a bacterial pathogen that affects more than half of the world’s population with gastro-intestinal diseases and is associated with gastric cancer. The cell surface of H. pylori is decorated with lipopolysaccharides (LPSs) composed of three distinct regions: a variable polysaccharide moiety (O-chain), a structurally conserved core oligosaccharide, and a lipid A region that anchors the LPS to the cell membrane. The O-chain of H. pylori LPS, exhibits unique oligosaccharide structures, such as Lewis (Le) antigens, similar to those present in the gastric mucosa and are involved in interactions with the host. Glucan, heptoglycan, and riban domains are present in the outer core region of some H. pylori LPSs. Amylose-like glycans and mannans are also constituents of some H. pylori strains, possibly co-expressed with LPSs. The complexity of H. pylori LPSs has hampered the establishment of accurate structure-function relationships in interactions with the host, and the design of carbohydrate-based therapeutics, such as vaccines. Carbohydrate microarrays are recent powerful and sensitive tools for studying carbohydrate antigens and, since their emergence, are providing insights into the function of carbohydrates and their involvement in pathogen-host interactions. The major goals of this thesis were the structural analysis of LPSs from H. pylori strains isolated from gastric biopsies of symptomatic Portuguese patients and the construction of a novel pathogen carbohydrate microarray of these LPSs (H. pylori LPS microarray) for interaction studies with proteins. LPSs were extracted from the cell surface of five H. pylori clinical isolates and one NCTC strain (26695) by phenol/water method, fractionated by size exclusion chromatography and analysed by gas chromatography coupled to mass spectrometry. The oligosaccharides released after mild acid treatment of the LPS were analysed by electrospray mass spectrometry. In addition to the conserved core oligosaccharide moieties, structural analyses revealed the presence of type-2 Lex and Ley antigens and N-acetyllactosamine (LacNAc) sequences, typically found in H. pylori strains. Also, the presence of O-6 linked glucose residues, particularly in LPSs from strains 2191 and NCTC 26695, pointed out to the expression of a 6-glucan. Other structural domains, namely ribans, composed of O-2 linked ribofuranose residues were observed in the LPS of most of H. pylori clinical isolates. For the LPS from strain 14382, large amounts of O-3 linked galactose units, pointing to the occurrence of a galactan, a domain recently identified in the LPS of another H. pylori strain. A particular feature to the LPSs from strains 2191 and CI-117 was the detection of large amounts of O-4 linked N-acetylglucosamine (GlcNAc) residues, suggesting the presence of chitin-like glycans, which to our knowledge have not been described for H. pylori strains. For the construction of the H. pylori LPS microarray, the structurally analysed LPSs, as well as LPS-derived oligosaccharide fractions, prepared as neoglycolipid (NGL) probes were noncovalently immobilized onto nitrocellulosecoated glass slides. These were printed together with NGLs of selected sequence defined oligosaccharides, bacterial LPSs and polysaccharides. The H. pylori LPS microarray was probed for recognition with carbohydratebinding proteins (CBPs) of known specificity. These included Le and blood group-related monoclonal antibodies (mAbs), plant lectins, a carbohydratebinding module (CBM) and the mammalian immune receptors DC-SIGN and Dectin-1. The analysis of these CBPs provided new information that complemented the structural analyses and was valuable in the quality control of the constructed microarray. Microarray analysis revealed the occurrence of type-2 Lex and Ley, but not type-1 Lea or Leb antigens, supporting the results obtained in the structural analysis. Furthermore, the H. pylori LPSs were recognised by DC-SIGN, a mammalian lectin known to interact with this bacterium through fucosylated Le epitopes expressed in its LPSs. The -fucose-specific lectin UEA-I, showed restricted binding to probes containing type-2 blood group H sequence and to the LPSs from strains CI-117 and 14382. The presence of H-type-2, as well Htype- 1 in the LPSs from these strains, was confirmed using specific mAbs. Although H-type-1 determinant has been reported for H. pylori LPSs, this is the first report of the presence of H-type-2 determinant. Microarray analysis also revealed that plant lectins known to bind 4-linked GlcNAc chitin oligosaccharide sequences bound H. pylori LPSs. STL, which exhibited restricted and strong binding to 4GlcNAc tri- and pentasaccharides, differentially recognised the LPS from the strain CI-117. The chitin sequences recognised in the LPS could be internal, as no binding was detected to this LPS with WGA, known to be specific for nonreducing terminal of 4GlcNAc sequence. Analyses of the H. pylori LPSs by SDS-PAGE and Western blot with STL provided further evidence for the presence of these novel domains in the O-chain region of this LPS. H. pylori LPS microarray was also applied to analysis of two human sera. The first was from a case infected with H. pylori (H. pylori+ CI-5) and the second was from a non-infected control.The analysis revealed a higher IgG-reactivity towards H. pylori LPSs in the H. pylori+ serum, than the control serum. A specific IgG response was observed to the LPS isolated from the CI-5 strain, which caused the infection. The present thesis has contributed to extension of current knowledge on chemical structures of LPS from H. pylori clinical isolates. Furthermore, the H. pylori LPS microarray constructed enabled the study of interactions with host proteins and showed promise as a tool in serological studies of H. pyloriinfected individuals. Thus, it is anticipated that the use of these complementary approaches may contribute to a better understanding of the molecular complexity of the LPSs and their role in pathogenesis.

Characterization of the methanol recovery process at Prio Biocombustíveis S. A.

In a industrial environment, to know the process one is working with is crucial to ensure its good functioning. In the present work, developed at Prio Biocombustíveis S.A. facilities, using process data, collected during the present work, and historical process data, the methanol recovery process was characterized, having started with the characterization of key process streams. Based on the information retrieved from the stream characterization, Aspen Plus® process simulation software was used to replicate the process and perform a sensitivity analysis with the objective of accessing the relative importance of certain key process variables (reflux/feed ratio, reflux temperature, reboiler outlet temperature, methanol, glycerol and water feed compositions). The work proceeded with the application of a set of statistical tools, starting with the Principal Components Analysis (PCA) from which the interactions between process variables and their contribution to the process variability was studied. Next, the Design of Experiments (DoE) was used to acquire experimental data and, with it, create a model for the water amount in the distillate. However, the necessary conditions to perform this method were not met and so it was abandoned. The Multiple Linear Regression method (MLR) was then used with the available data, creating several empiric models for the water at distillate, the one with the highest fit having a R2 equal to 92.93% and AARD equal to 19.44%. Despite the AARD still being relatively high, the model is still adequate to make fast estimates of the distillate’s quality. As for fouling, its presence has been noticed many times during this work. Not being possible to directly measure the fouling, the reboiler inlet steam pressure was used as an indicator of the fouling growth and its growth variation with the amount of Used Cooking Oil incorporated in the whole process. Comparing the steam cost associated to the reboiler’s operation when fouling is low (1.5 bar of steam pressure) and when fouling is high (reboiler’s steam pressure of 3 bar), an increase of about 58% occurs when the fouling increases.