High Performance Liquid Chromatography (HPLC) was used to investigate the utility of this technique for the analytical and preparative separation of components of aquatic fulvic acids (FA). Three modes of HPLC namely adsorption, anion exchange and reversed phase were evaluated. Aquatic fulvic acids were either extracted from surface water and sediment samples collected from the Southwest of the U.S., or were provided in a high purity form from the USGS. On the adsorption mode, a major fraction of aquatic fulvic acid was isolated on a semipreparative scale and subjected to Carbon-13 NMR and FAB Mass Spectroscopy. Results indicated that (1) The analyzed fraction of fulvic acid contains more aliphatic than aromatic moieties; (2) Methoxy, carboxylic acids, and esters are well-defined moieties of the macromolecule; (3) Phenolic components of the macromolecules were not detected in the Carbon-13 NMR spectrum possibly because of the presence of stable free radicals. Results of the anion exchange mode have shown that at least three types of acidic functionalities in aquatic fulvic acid can be separated. Results also indicated that aquatic fulvic acid can be progressively fractionated by using subsequent modes of HPLC. Results of reversed phase mode have shown that (1) The fractionation of aquatic fulvic acid by RP-HPLC is essentially controlled by the polarity and/or pH of the carrier solvent system; (2) Under different RP-HPLC conditions aquatic fulvic acid from several locations are fractionated into the same major components; (3) Fulvic acid extracted from water and sediment from the same site are more similar than those extracted from different sites; (4) Cationic and anionic ion pair reagents indicated the presence of amphoteric compounds within the polymeric structure of fulvic acid. Each mode of HPLC provided a characteristic profile of fulvic acid. The results of this research provided basic information on the behavior of aquatic fulvic acids under three modes of HPLC. Such informations are prerequisite for further investigation by spectroscopic methods.

The preparation of layered double hydroxides via co-precipitation of a divalent/trivalent metal solution against a base results in 1 mm LDH particles with a disorganized metal lattice. Research was performed to address these morphological issues using techniques such as Ostwald ripening and precipitation via aluminate. Another interesting issue in layered double hydroxide materials is the uptake and orientation of anions into the interlayer. Questions about iron cyanide interlayer anions have been posed. Fourier transform infared spectroscopy and powder x-ray diffraction have been used to investigate these topics. It was found that factors such as orientation, anion charge, and anion structure depended on the divalent/trivalent metal ratio of the hydroxide layer and reactivity time. The cyanide self-addition reaction is an important reaction of classical prebiotic chemistry. This reaction has been shown to give rise to amino acids, purines and pyrimidines. At cyanide concentrations similar to that expected on the early earth, hydrolysis to formamide rather than self-addition occurs. One theory to alleviate this side reaction is the use of minerals or clays that are thought to concentrate and catalyze prebiotics of interest. Layered double hydroxides have been studied as a catalyst for this reaction.

Size selective reactivity has been observed in pure aluminum cluster anions as a result of Lewis acid and base pairs. Using this a starting point, the goal of this study has been to explore how reactivity is affected with the addition of one or more ligand, which may induce active sites on the surface of the metal clusters. To study this, a theoretical investigation was undertaken on Al13Ix- and Al14Iy- (x=0-2, y=2-4) and their reactivity with methanol. The hypothesis was that iodine can induce a Lewis base site on the opposite side of the cluster, which may enhance reactivity. In results that are consistent with preliminary experimental data, it was found that the Al13Ix- series has a large energy barrier with respect to the cleavage of the O-H bond of methanol. The clusters of the series act as an extremely poor Lewis acids, and as a result, these clusters are relatively inert to methanol etching. On the other hand, the Al14Iy- series has a low barrier and is expected to react rapidly with methanol. The series is found to be most reactive at an aluminum adatom that is bound to an iodine due to the iodine extracting charge from the aluminum cluster creating a strong Lewis acid site.

Water-soluble deep-cavity cavitands provide a rare opportunity to study self-assembly driven by the hydrophobic effect. These molecular hosts dimerize in the presence of certain guest molecules to form water-soluble molecular capsules. These systems have given rise to numerous novel chemical phenomena and have potential use in drug delivery. The host octaacid (OA) has been particularly well-characterized, but studies are limited to basic pH because of limited host solubility. Herein we report an improved synthesis of OA and the syntheses of three new water-soluble deep-cavity cavitands. The new hosts are soluble at neutral pH, increasing relevance for biological studies. The new syntheses are versatile enough to apply to the synthesis of additional water- soluble cavitands in the future. We also describe preliminary characterization of the molecular recognition properties of the new hosts. Binding of organic guest molecules to form 1:1 host:guest complexes and 2:1 host:guest capsules was qualitatively similar to that of OA. However, binding of anions spanning the Hofmeister series revealed interesting new behavior. The new hosts bound a wider range of anions inside the hydrophobic pocket with much higher association constants. Moreover, external binding of several anions to the cavitand pendant feet was observed. Looking towards biological applications, we desired to learn how these molecules interact with phospholipid membranes. Six water-soluble cavitands were tested for their ability to permeabilize liposomal POPC membranes. One host showed very high potency in permeabilizing membranes, while three other hosts showed moderate activity. Host binding of POPC was found to be at least one factor in host-induced permeabilization. A requenching assay to determine leakage mechanism strongly supported all-or-none leakage, whereby some vesicles lose all contents while others lose none. These results suggest that these cavitands induce partial transient leakage of vesicles by the formation of transient membrane pores. These findings show potential for the use of these hosts as drug delivery carriers, antimicrobial compounds, and tools in membrane alteration studies.

A new “best match” match model has been developed to account for adduct formation on multiply charged peptides observed in negative ion electrospray mass spectrometry. To obtain a stable adduct, the model necessitates an approximate matching of apparent gas-phase basicity (GBapp) of a given proton bearing site on the peptide with the gas-phase basicity (GB) of the anion attaching at that site. Evidence supporting the model is derived from the fact that singly charged adducts were only observed for lower GB anions: HSO4-, I-, CF3COO-. Ions that have medium GBs (NO3-, Br-, H2PO4-) only form adducts having -2 charge states, whereas Cl- (higher GB) can form adducts having -3 charge states. Hydrogen bonds are the main interactions pertinent to the “Best Match” model, however, ion-ion interactions formed between peptides ([Glu]Fibrinopeptide B, Angiotensin I or [Asn1,Val5]-Angiotensin II) and low GB anions (ClO4- or HSO4-) have been established by CID-MS/MS. Evidence for ion-ion interactions comes especially from product ions formed during the first dissociation step, where, in addition to the expected loss of the anion or neutral acid, other product ions that require covalent bond cleavage (i.e., H2O or NH3 loss) are also observed. In this study, the “Best Match” model is further supported by the decomposition behavior of adducts formed when Na+/H+ exchange has occurred on peptides. Na+/H+ exchanges were found to occur preferentially at higher acidity sites. Without any Na+/H+ exchange, F- and CH3COO- can hardly form observable adducts with [Glu]Fibrinopeptide B. However, after multiple Na+/H+ exchanges, F- and CH3COO- do form stable adducts. This phenomenon can be rationalized by considering that Na+ cations serve to “block” the highly acidic sites, thereby forcing them to remain overall neutral. This leaves the less acidic protons available to match with higher GB anions. According to the "best match" model, high GB anions will match with high GBapp sites on the peptide, whereas low GB anions will match with low GBapp peptide sites. High charge states readily augment GBapp of the peptide (through-space effect). Na+/H+ exchanges substantially decrease GBapp by neutralizing charged sites, while slightly increasing intrinsic GBs by the inductive effect.

Ziel der Dissertation „Neue künstliche Guanidiniocarbonylpyrrol-Rezeptoren zur Komplexierung von Oxo-Anionen in Wasser“ war die Weiterentwicklung dieser Rezeptoren nach Schmuck für die Komplexierung insbesondere von Carboxylaten, um sie hinsichtlich Bindungsaffinität und Substratspezifität zu optimieren. Dazu wurde zunächst die Synthese zweier wichtiger Grundbausteine in einzelnen Schritten vollständig überarbeitet, wobei veränderte Reaktionsbedingungen und Aufarbeitungsschritte zu gesteigerten Ausbeuten führten. Dadurch ist es nun möglich, diese Bausteine effizienter zu synthetisieren und im Multigramm-Maßstab für die Darstellung von Rezeptoren zur Oxo-Anionen-Erkennung einzusetzen. Weiterhin wurde die Verbesserung der Komplexierungseigenschaften gegenüber Carboxylaten auf zwei Arten untersucht: zum einen durch das Anbringen eines zusätzlichen Seitenarms an der Guanidinio-Einheit zur Bildung von Guanidiniocarbonylpyrrol-Tweezer-Rezeptoren, zum anderen durch das Einführen einer zweiten positiven Ladung neben der Carboxylat-Bindungsstelle (CBS) zur Darstellung biskationischer Guanidiniocarbonylpyrrol-Rezeptoren. Zur Darstellung von Tweezer-Rezeptoren wurde ein zusätzlicher Seitenarm an der N’-Position der Guanidinio-Einheit angebracht. Die beiden Arme sollten ein Substrat pinzettenartig von zwei Seiten, mit der CBS als Kopfgruppe, komplexieren können. Durch zusätzliche Wechselwirkungen des neuen Seitenarms sollte neben einer stärkeren Komplexierung vor allem eine höhere Substratspezifität erzielt werden. Die experimentell ermittelten Bindungskonstanten lagen allerdings im Bereich der N’-unsubstituierten Rezeptoren. Somit stellen die Tweezer-Modifikationen daher keine Verbesserung der Guanidiniocarbonylpyrrol-Rezeptoren dar. In einem weiteren Projekt zur Rezeptor-Optimierung wurden, durch Einführung einer zweiten positiven Ladung in Form einer terminalen Ammonium-Gruppe, biskationische Guanidiniocarbonylpyrrol-Rezeptoren erfolgreich synthetisiert. Die Komplexierungseigenschaften dieser biskationischen Rezeptoren wurden in Bindungsstudien vornehmlich mit Aminosäurecarboxylaten mittels UV- und Fluoreszenz-Spektroskopie, Massenspektrometrie, NMR-Spektroskopie, ITC und Molecular Modeling Berechnungen untersucht. Anhand der Substratspezifität der biskationischen Rezeptoren wurde deutlich, dass die Spacerlänge, an der die zusätzliche positive Ladung angebracht ist, eine entscheidende Rolle bei der Komplexierung spielte. Galten eigentlich starre, präorganisierte, kurze Linker als vorteilhaft hinsichtlich der Entropie, so ist hier zu erkennen, dass längere, flexiblere Linker zu einer besseren Komplexierung führen können, wenn geeignete zusätzliche nichtkovalente Wechselwirkungen möglich sind. Die biskationischen Rezeptoren stellen damit eine Optimierung des Carboxylat-Bindungsmotivs der Guanidiniocarbonylpyrrol-Rezeptoren nach Schmuck in der Anionen-Erkennung dar. / The main focus of the thesis “New artificial guanidiniocarbonyl pyrrole receptors for the complexation of oxo-anions in water” is the optimization of these receptors introduced by Schmuck for the oxo-anion recognition, especially carboxylates, in aqueous solution. Therefore, the details of the synthesis of two important building blocks were completely revised and yields increased by changing reaction conditions, workup and isolation steps. The new optimized, facile and efficient synthetic route to these N-protected guanidinocarbonyl pyrrole derivatives allows now a multi-gram synthesis of these versatile compounds as needed for the synthesis for a variety of supramolecular anion binding motifs. Furthermore two strategies to optimize the anion recognition have been pursued: On the one hand tweezer receptors were developed by connecting an additional side chain to the head group at the N’-position of the guanidino group. On the other hand another second positive charge was introduced into the receptor besides the carboxylate binding site (CBS) by an ammonium group to get bis-cationic receptors The tweezer receptors were developed in order to complex the guest from more than one side. A second side chain, connected to the N’-position of the guanidinio motif, leads to this kind of receptor, with the CBS as a head group. By using additional non covalent interactions in both side chains, the association constant as well as the specificity should be increased. In case of the optimization of the binding properties, the tweezer receptors have showen binding properties similar to the N’-unsubstituted guanidiniocarbonyl pyrrole receptors with respect to association constants and specificity. For this reason the tweezer receptors were no improvement of the guanidiniocarbonyl pyrrole receptors. In another project for optimization of the binding properties several bis-cations were synthesized, with a simple primary ammonium cation attached via flexible linkers of varying length to a guanidiniocarbonyl pyrrole. In UV-binding studies in aqueous buffer these bis-cations have shown efficient binding of various N-acetyl amino acid carboxylates. Further investigations by fluorescence spectroscopy, mass spectrometry, NMR spectroscopy, ITC as well as molecular mechanics calculations confirm the complexation by participation of the second charge in the complexation of the carboxylic function and therefore an increasing in complex stability. It is generally assumed, that short and rigid linkers are better for complexation due to the entropy and that a steady decrease of a linker length increase the complex stability. However, this case shows, that complex stability can increase while increasing the flexibility of a linker until other non covalent interactions are possible. Hence, the bis-cationic receptors indeed represent an optimisation of the guanidiniocarbonyl pyrrole receptors for oxo-anion-binding.

O efeito do balanço cátion-aniônico da dieta (BCAD) no desempenho, balanço macromineral (cálcio, fósforo, sódio e potássio), na fermentação ruminal , pH urinário e na concentração sérica de cálcio, foi estudado utilizando-se 25 ovinos machos, da raça Santa Inês, por um período de 75 dias. O delineamento experimental foi em blocos ao acaso, com cinco tratamentos. Para a manipulação do BCAD foram adicionados sulfato de amônio e bicarbonato de sódio, obtendo-se os seguintes tratamentos: -160, -40, 140, 250 e 500 mEq/kg MS. O aumento do BCAD resultou em aumento da IMS, aumento em ganhos diários e eficiência alimentar (P<0,05). O pH ruminal, pH urinário e o volume urinário aumentaram com aumento do BCAD (P<0,05). O perfil de ácidos graxos ruminais não foi afetado pelo BCAD. O BCAD influenciou o metabolismo macromineral principalmente o de cálcio, com a diminução do BCAD o cálcio sérico aumentou. Dietas aniônicas aumentaram a excreção urinária de cálcio, entretanto a retenção não foi afetada. / The dietary cation-anion balance (DCAB) effect in performance, macromineral (calcium, phosphorus and sodium) balance, urinary pH and calcium serum concentration, was studied in 25 males sheeps, from Santa Ines breed, during 75 days. The design used was randomized complete blocks, with five treatments. For DCAB manipulation were added ammonium sulfate and sodium bicarbonate in order to achieve the values: -160, -40, 140, 250 e 500 meq/kg DM. Increasing the DCAB resulted in higher DMI, daily gains and feed efficiency (P<0,05). Rumen pH, urine pH and urine volume increased as DCAB increased (p<0,05). Ruminal VFA profile was not affected by DCAB. The DCAB affected the calcium metabolism, decresing the DCAB the serum calcium increased. Dietary anionic increased urinary excretion of calcium, however calcium retention was not effect.

O Brasil possui uma imensa diversidade biológica, na qual muitos compostos bioativos podem ser encontrados e utilizados em beneficio à sociedade. No entanto, processos de degradação do ambiente e introdução de espécies exóticas têm contribuído ao conhecimento e uso limitadosde muitas plantas nativas, sendopequena a quantidade de estudos sobre sua composição química e potencial biológico. A prevenção de doenças crônicas constantemente vem sendo associada à atividade antioxidante de metabólitos secundários dos vegetais, principalmente os fenólicos. Assim, o objetivo deste trabalho foi avaliar a atividade antioxidante e composição fenólica de cinco espécies frutíferas nativas do Brasil (G. brasiliensis, E. leitonii, E. involucrata, E. brasiliensis e E. myrcianthes). Os métodos capacidade de redução do Folin-Ciocalteau, autoxidação do sistema ?-caroteno/ácido linoleico, capacidade de absorção de radicais oxigênio (ORAC), sequestro dos radicais livres DPPH e ânion superóxido foram aplicados na determinação da atividade antioxidante dos extratos etanólicos de folha, casca, semente e polpa das espécies selecionadas. As amostras demonstraram significativa atividade antioxidante e, em alguns casos, superior as frutas comumente consumidas pela população brasileira. Em geral, folhas apresentaram as maiores atividades, mas o destaque foi a semente de E. leitonii que exibiu os melhores resultados em quatro dos cinco métodos utilizados: 120,67 mg AG.g-1 na redução do reagente Folin; 7,08 Gmol Trolox.g-1 no ?-caroteno; EC50 de 0,26 mg.mL-1 e 0,07 mg.mL-1 no sequestro do ânion superóxido e DPPH, respectivamente; 514,75 Gmol Trolox.g-1 no ORAC para o qual a folha de E. involucrata obteve o maior valor (1393,3 Gmol Trolox.g-1). Os extratos das espécies nativas também demonstram efeito antioxidante contra radicais de relevância biológica, como peroxila e superóxido. Por meio de CG-EM e CLAE acoplado a arranjo de fotodiodos, os principais compostos fenólicos encontrados nos extratos vegetais foram catequina, epicatequina e ácido gálico. Este trabalho demonstrou o grande potencial antioxidante das frutíferas nativas brasileiras, evidenciando assim possível efeito positivo em sistemas biológicos. / Brazil has a great biodiversity, in which many bioactive compounds can be found and used to benefit the society. However, environmental degradation processes and introduction of exotic species have contributed to limited use and knowledge of many native plants, reflecting in few studies about chemical composition and biological potential. The prevention of chronic diseases has been constantly associated with the antioxidant activity of plants secondary metabolites, mainly the phenolics. The objective of this study was to evaluate the antioxidant activity and the phenolic composition of five Brazilian native fruitful species (G. brasiliensis, E. leitonii, E. involucrata, E. brasiliensis e E. myrcianthes). The methods of Folin-Ciocalteau reducing capacity, co-oxidation of ?-carotene/linoleic acid system, oxygen radical absorbance capacity (ORAC), DPPH and superoxide free radical scavenging were used to determine the antioxidant activity of ethanolic extracts of leaf, peel, seed and pulp of the selected species. The samples showed significant antioxidant activity and, in some cases, it was superior to fruits commonly consumed by Brazilian population. In general, leaves presented the highest activities, but the seed of E. Leitonii stood out exhibiting the best results in four of the five methods: 120.67 mg GA.g-1 in the Folin reducing; 7.08 Gmol Trolox.g-1 in the ?-carotene; EC50 of 0.26 mg.mL-1 and 0.07 mg.mL-1 in the superoxide and DPPH scavenging, respectively; 514.75 Gmol Trolox.g-1 in the ORAC, for which the E. Involucrata leaf had the highest value (1393.3 GmolTrolox.g-1). The extracts of native species also demonstrate antioxidant effect against radicals of biological relevance, such as peroxyl and superoxide. By GC-MS and HPLC coupled to a photodiode array, the major phenolic compounds found in the plant extracts were catechin, epicatechin and gallic acid. In this study, Brazilian native fruitful presented high antioxidant potential, showing a possible positive effect on biological systems.

Os efeitos do balanço cátion-aniônico da dieta (BCAD) na degradabilidade ruminal da MS e PB do farelo de soja, degradabilidade ruminal da MS e amido do fubá de milho, foram estudados em dois experimentos com diferentes proporções de volumoso:concentrado (experimento 1 - 60:40; experimento 2 - 40:60), em 12 bezerros Holandeses (peso médio de 91,55kg e de 117,62kg respectivamente), portadores de cânulas ruminais, realizados no Campus Administrativo de Pirassununga/SP, na FZEA/USP. Os períodos experimentais foram de 35 dias, sendo 14 dias de adaptação dos animais às gaiolas e às dietas e 21 dias de colheita de dados, com 15 dias de descanso. O delineamento foi o inteiramente casualizado, com três tratamentos e quatro repetições em ambos os experimentos. Para a manipulação do BCAD, foram adicionados sulfato de amônio e bicarbonato de sódio, obtendo-se os seguintes tratamentos no experimento 1: -123, +218 e +341 mEq/kg MS; e no experimento 2: -127, +207 e +397 mEq/kg MS. Utilizou-se silagem de milho como fonte de volumoso. Os níveis de BCAD influenciaram as degradabilidades ruminais da MS e PB do farelo de soja com a proporção de volumoso:concentrado de 60:40 (P<0,10). As degradabilidades ruminais da MS e amido do fubá de milho com a proporção volumoso:concentrado de 60:40, não foram afetadas pelos níveis de BCAD (P>0,10), assim como as degradabilidades ruminais da MS e PB do farelo de soja, MS e amido do fubá de milho com a proporção volumoso:concentrado de 40:60 (P>0,10). / The DCAB effects on dry matter (DM) and crude protein (CP) degradability of soybean meal and DCAB effects on DM and corn meal degradability were investigated in two trials with different rate of roughage:concentrate (experiment 1: 60:40, experiment 2: 40:60). Twelve Holstein calves with ruminal cannula were used in each experiment (91,55 kg and 117,62 kg of mean weight, respectively). Both experiments were conducted at FZEA/USP, Pirassununga/SP. The experimental periods were constituted by 35 days (14 days adaptation and 21 days data collection) with 15 days of interval. It was used completely design, with three treatments and four repetitions. For DCAB manipulation were added ammonium sulfate and sodium bicarbonate in order to achieve the following values: experiment 1: -123, +218, +341 mEq/kg DM, experiment 2: -127, +207, +397 mEq/kg DM. The diet was based on corn silage as forage. The DCAB ratios affected DM and soybean meal CP degradabilities when 60:40 roughage:concentrate ratio were used (P<0,10). DM and corn meal degradabilities were not affected by DCAB ratios (P>0,10), as well as DM and soybean meal CP degradabilities when 40:60 roughage:concentrate ratio were used P<0,10).

A via de amplificação da secreção de insulina estimulada por glicose (GSIS) é um fenômeno discutido na literatura, cujos componentes são amplamente debatidos. Evidências sugerem que a condutância a Cl- compõe parte desta via. Porém, o mecanismo pelo qual essa condutância desempenharia papel na via de amplificação ainda é debatido, e, além disso, as ferramentas farmacológicas para estudo dessas afeta o transporte de outros ânions, como bicarbonato (HCO3-). Buscamos neste trabalho compreender a contribuição do transporte desses ânions para a via de amplificação da GSIS levando em consideração a distribuição de Cl- e HCO3- extracelular em células INS-1E. Concluímos que o transporte de ânions nas células INS-1E não contribui para a via de amplificação da GSIS, porém essas células não expressaram os canais CFTR e Anoctamina 1 que foram relacionados com esse fenômeno. Acreditamos que em células secretoras de insulina que expressem esses canais, o transporte de ânions possua alguma relevância funcional. / The amplification pathway of glucose stimulated insulin secretion (GSIS) is a phenomenon discussed in the literature, which components are broadly debated. Evidence suggests that Cl- conductance composes part of this pathway. However, the mechanism that this conductance would play role on the amplification pathway still is debated, and, besides that, the pharmacological tools to study these affects transport of other anions, such as bicarbonate (HCO3-). We aimed in this study to understand the contribuition of anion transport for the amplification of GSIS considering the Cl- and HCO3- extracellular distribuition in INS-1E cells. We concluded that anion transport in INS-1E cell line do not contribute for the amplification pathway of GSIS, however those cells do not express CFTR and Anoctamin 1 channels which were related with this phenomenon. We believe that in insulin secretin cells that express those channels, the anion transport may have a functional relevance.