Synthesis of charged cyclodextrin highly soluble in organic solvents was made by exchanging the inorganic counter ion (Na+) of heptakis (2,3-di-Omethyl- 6-O-sulfo)-β-CD (Na7HDMS) with tetrabutylammonium (TBA+), to produce TBA7HDMS. The same ion exchange was used to synthesize the TBA salts of the analogous CDs TBA6HxDMS and TBA8ODMS. Indirect-UV detection capillary electrophoresis (CE) and 1H NMR were used as the characterization methods. Separations of thirteen pharmaceuticals were made using TBA7HDMS as the chiral resolving agent in aqueous CE. On the other hand, a set of twenty pharmaceuticals was used for the enantiomer separations in non-aqueous CE (NACE). Comparison between the results obtained with TBA7HDMS in aqueous and non-aqueous CE were made. In addition, comparison between the results obtained with TBA7HDMS and Na7HDMS in aqueous and non-aqueous CE were made as well.

The isolation of individual enantiomers of drugs is an important subject of interest in the pharmaceutical and medical fields, because stereochemistry can have a significant effect on the biological activity of the drug. Therefore, it is important to develop enantiomeric separation methods for the determination of the optical purity of drugs, since the undesired enantiomer is regarded as one of the impurities. The available single isomer anionic cyclodextrins (CD) can resolve the enantiomers of only a few weakly acidic analytes. To rectify this problem, the chloride salts of heptakis(6-deoxy-6-morpholinio)-cyclomaltoheptaose (HMBCD), and mono(6- deoxy-6-N,N,N r,N r,N r-pentamethylethylenediammonio)-cyclomaltoheptaose (PEMEDA-BCD), the first members of the permanently charged, single-isomer cationic cyclodextrin family, have been synthesized. The purity of process intermediates and final products was determined by HPLC-ELSD and indirect UV-detection capillary electrophoresis. Structural identity was verified by 1D and 2D NMR and massBoth cationic CD derivatives have been used for the separation of the enantiomers of strong acid, weak acid, weak base, ampholytic, and neutral analytes by capillary electrophoresis. Because the charge state of these cationic chiral resolving agents is independent of the pH of the buffer, separation could be performed in both low and high pH buffers without compromising the charge density of the resolving agent. Contrary to expectation, the multiply charged HMBCD showed poor complexation with the newly synthesized strong electrolyte test analytes. The weak binding between the analytes and HMBCD resulted in separation of enantiomers of only three strong electrolyte analytes. Strong complexation was observed between PEMEDA-BCD and the anionic and nonionic analytes in both low and high pH buffers, though complexation was stronger in the high pH buffer. Due to strong complexation between the anions and PEMEDA-BCD, only low concentrations of the resolving agent were required to effect good enantiomer resolutions. spectrometry.

As the major protein of the lens, α-crystallin is a molecular chaperone that stabilises lens proteins to prevent their precipitation into solution. In this role it is vital in maintaining lens transparency. The chaperone ability of α-crystallin and its individual subunits, αΑ- and αB-crystallin, has been shown to be sensitive to a variety of environmental and intrinsic factors, including temperature, denaturation and post-translational modification. The effect of pH on α-crystallin chaperone ability, however, has not been thoroughly investigated. There is limited evidence to suggest that the chaperone ability of α-crystallin is pH-sensitive such that α- crystallin is a significantly worse chaperone at pH 6.0 than at pH 8.0. This is of physiological significance since in the lens there is a measurable pH gradient of pH 7.2 in outer lens cells, compared to pH 6.7 in the lens nucleus. A loss of α-crystallin chaperone function in the lens nucleus, as a consequence of decreased pH, may compromise lens transparency. Similarly, extra-lenticular fibrillar aggregation of some disease-related target proteins (Aβ-peptide, for example) is promoted by acidic pH. This study investigates the effect of pH on the chaperone ability of α-crystallin and its subunits. Further, this study characterises the structural changes to α-crystallin accompanying pH variation in an attempt to explain the structural basis for the observed pH sensitivity. In addition, this study examines the chaperone function of cyclodextrins, a class of chemical chaperones that may act in conjunction with α-crystallin as part of a two-step protein refolding pathway. This study demonstrated that the chaperone activity of α-crystallin is pH sensitive between pH 6.0 and 8.0; the ability of α-crystallin to protect against temperature- and reduction-stress induced amorphous aggregation is significantly reduced at pH 6.0 and 6.5 compared to pH 7.0 and above. The decreased chaperone ability of α-crystallin at pH 6.0 and 6.5 was accompanied by partial unfolding of the protein, and a loss of secondary structure, while α-crystallin quaternary structure remained unchanged. Interestingly, α-crystallin was found to have significant chaperone ability below pH 4.0, conditions under which α-crystallin is largely unfolded. The unfolding of α-crystallin at pH 6.0 and 6.5 is comparatively minor, and it is difficult to say whether this unfolding is directly responsible for the observed pH sensitivity of α-crystallin chaperone ability. The thermal stability of α-crystallin was compromised at pH 6.0 and 6.5, which may partially explain its decreased chaperone ability at these pH values in heat-stress assays conducted at temperatures above 50oC. However, α-crystallin chaperone activity remained pH sensitive at 37°C and 45°C, at which temperatures it is thermally stable. Blocking exposed αB-crystallin histidine residues by chemical modification removed, to a large extent, the pH-sensitivity of its chaperone activity. This suggests that the protonation of an exposed histidine residue(s) at pH 6.0 and 6.5 is responsible for the observed pH sensitivity of α-crystallin chaperone ability. Inhibiting the protonation of a specific histidine residue, H83, by site-directed mutagenesis (H83A) did not remove the pH sensitivity of αB-crystallin chaperone activity, and suggests that protonation of this residue alone does not explain the decreased chaperone ability of α-crystallin at mildly acidic pH. This residue lies within the putative chaperone-binding region of αB-crystallin, and is highly conserved between species and between the human small heat shock proteins. It appears that the protonation of several histidine residues, or residues other than H83, is primarily responsible for the influence of pH on α-crystallin chaperone ability observed in this study. The observed decrease in α-crystallin chaperone function below pH 7.0 partially explains the preferential formation of age-related cataract in the lens nucleus, as the chaperone ability of α-crystallin would be compromised under the mildly acidic conditions characteristic of the nucleus. Additionally, the pH sensitivity of α-crystallin chaperone ability may be significant in the ability of extra-lenticular αB-crystallin to inhibit amyloid-related disease at sites of localised acidosis. Cyclodextrins are a family of cyclic oligosaccharides that have been shown to function as chemical chaperones under specific protein aggregation conditions. Cyclodextrins have been demonstrated to facilitate the refolding of chemicallystressed target proteins that have already bound to synthetic nanogels, which act in a manner reminiscent of small heat shock proteins. In this study, cyclodextrins were unable to act in conjunction with α-crystallin to facilitate the refolding of thermallystressed target proteins. β-Cyclodextrin (βCD) demonstrated little or no ability to inhibit the amorphous aggregation of target proteins, but was able to significantly inhibit the fibrillar aggregation of a number of target proteins, including the diseaserelated A53T α-synuclein mutant. Characterisation of the binding of βCD to target proteins during fibrillar aggregation via circular dichroism, intrinsic and extrinsic fluorescence and competitive chaperone assays provided a model of the cyclodextrin chaperone mechanism. In this model, cyclodextrins interact with already partially unfolded, pre-fibrillar protein intermediates via the insertion of aromatic residues into the cyclodextrin anulus, and by doing so inhibit intra-fibrillar π-bonding and protofilament assembly. This suggests the potential for cyclodextrins as therapeutic molecular chaperones in vivo that may be able to inhibit the pathogenic aggregation of target proteins. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1368102 / Thesis (Ph.D.) - University of Adelaide, School of Chemistry and Physics, 2009

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Resumo: Este trabalho tem como objetivo estudar a produção da enzima ciclodextrina glicosiltransferase (CGTase) empregando culturas de Bacillus sp subgrupo alcalophilus utilizando como fonte de carbono fécula de mandioca (polvilho) proveniente de uma fecularia de mandioca. Nos ensaios foram empregados Bacillus sp subgrupo alcalophilus, isolado de água residuária de uma fecularia de mandioca. Para avaliar a produção de CGTase mediante a atividade enzimática pelo Bacillus sp subgrupo alcalophilus foi utilizado um planejamento fatorial a dois (2) níveis, estudando como variáveis as concentrações da fonte de carbono (polvilho), de nitrogênio e de carbonato de sódio. Os experimentos foram realizados em Erlenmeyers de 300 mL de capacidade contendo 100 mL do meio de produção com pH inicial de 9,2, a 150 rpm e temperatura de 35 ± 1ºC durante 72 horas de fermentação. A produção de CGTase foi monitorada pela determinação da atividade enzimática (U/mL). Após os ensaios realizados em frascos foram realizados experimentos em biorreator de 5 L de volume útil, contendo 2 L do meio de produção, pH de 9,20, agitação de 150 rpm e temperatura de 35 ± 1ºC durante 72 h de fermentação, utilizando um planejamento fatorial a dois (2) níveis, estudando como variáveis as taxas de aeração e a velocidade de agitação. A otimização das concentrações da fonte de carbono, nitrogênio e carbonato de sódio foram obtidas a partir de um planejamento experimental composto central (PCC) e seus resultados analisados pelas superfícies de resposta. Os melhores resultados do planejamento encontrados no ponto central, corresponderam a 6,96 g/L da fonte de carbono, 8,07 g/L de nitrogênio e 9,45 g/L de carbonato de sódio. A maior produtividade obtida de CGTase após 72 horas de fermentação, foi 98,86 U/mL com valor teórico de 98,87 U/mL. A partir do melhor resultado obtido no PCC, determinou-se... (Resumo completo, clicar acesso eletrônico abaixo) / Abstract: The aim of this study was to investigate the Cyclodextrin glycosyltransferase (CGTase) enzyme production by Bacillus sp subgroup alcalophilus using cassava starch (manioc flour) as a carbon source. Bacillus sp subgroup alcalophilus was isolated from wastewater of cassava flour industry. To evaluate the assays results, two (2) levels of complete factorial experimental design was used, studying the variables: carbon source, nitrogen and sodium carbonate concentrations. The experiments were performed in 300 mL erlenmeyer flasks containing 100 mL of medium production with initial pH of 9.2, at 150 rpm and 35±1ºC, during 72 hour. CGTase production was monitored by measurements of enzymatic activity (U/mL). After the flasks experiments, assays was running in 5 L containing 2 L of medium production, pH 9.2, 35 ± 1ºC during 72-hours using a factorial design at two (2) levels for aeration and agitation rate. The optimization of carbon source, nitrogen and sodium carbonate concentrations was obtained by a central composite design and their results analyzed by surface response. The best results was located on the central point, 6.96 g/L of carbon source, 8.07 g/L of nitrogen and 9.45 g/L of sodium carbonate. The CGTase activity predicted by model was 98.86 U/mL and the experimental activity obtained was 98,87 U/mL. After the best results obtained by PCC, the conditions of aeration (vvm) and agitation (rpm) rates was determined in bioreactor using a complete factorial experimental design. The best conditions, of 2.18 vvm and agitation of 157.07 rpm gave a experimental predicted CGTase activity of 130.36 U/mL, which was very close to the theoric CGTase activity of 130.33 U/mL. / Orientador: Antonio Carlos Simões Pião / Coorientador: Jonas Contiero / Banca: Heloiza Ferreira Alves do Prado / Banca: Sandra Regina Ceccato Antonini / Mestre

Cyclodextrins (CDs) are macrocycles composed of several glucose units bound through α-1,4 glycosidic linkages. They can be chemically modified to display functional groups on their primary or secondary rim. CDs display these groups in defined geometries ideally suited to bind biomacromolecules. Moreover, CDs have a hydrophobic cavity that allows them to form stable host-guest complexes with lipophilic molecules. This combination of functionality and guest binding ability makes CDs important scaffolds for the design of functional supramolecular systems. This thesis explored the interaction of heptakis-[6-deoxy-6-(2-aminoethylsulfanyl)]-β-cyclodextrin (1) with many hydrophobic guest molecules. The binding constants of CD host-guest interactions were measured using ITC and fluorometry-based approaches. These studies revealed 1 to form the highest affinity 1:1 cyclodextrin-guest complexes reported to date. This thesis then explored the use of CD inclusion complexes as biomacromolecular sensors. CD 1 and its derivatives were used to develop self-assembling sensors. First, a library of polycationic CDs with differing charge distribution was synthesized. The sensing motif was synthesized by covalently linking a quinolinium fluorophore to lithocholic acid (LCA). The CD-based binding motifs and the LCA-based sensing motif self-assemble through host-guest interactions (i.e. 1 binding to LCA displays a Ka = 5.52 × 107 M-1). These inclusion complexes were then used as an array of self-assembling sensors capable of differentiating between pure and contaminated samples of heparin (anticoagulant). To capitalize on the promise of CD 1 a new technique was explored to functionalize a single amine of 1. The technique relies on an S to N acyl transfer from a guest molecule to a CD host resulting in the mono-acylation of the host. The importance of the linker between the guest and the reactive acylating agent was fully explored. Furthermore, two CD probes are synthesized and are shown to display differential fluorescent responses with a small series of proteins.

Triazenes are molecules characterized by having the triazeno group (–N=N–N<). They are known as photoactive molecules due to trans-cis photoinduced isomerization around the N=N bond. Their nucleophilic character makes them good ligands to metal centers. A study on the potential ability of photochromic organic ligand triazenes to undergo reversible cis-trans photoisomerization and, in turn, photoreversible complexation with metal cations was carried out in aqueous solutions. Metal-triazenide complexes are instantaneously formed upon addition of metal cations to trans-triazenes dissolved in aqueous solutions. For silver-triazenide and mercury-triazenide complexes, the metal-to-ligand ratios are 1:1 and 1:2, respectively. Unfortunately, target metal-triazenide complexes do not photoisomerize upon 355 nm laser excitation. Triazenes are also known to be unstable materials under acidic conditions. A study on the effects of cyclodextrins (CDs) on the rate of acid-catalyzed decomposition of 1,3-diphenyl-triazenes was carried out in 2% MeOH aqueous buffer solutions by means of spectroscopic methods. CDs inhibit triazenes decomposition through inclusion complex formation. The inclusion complexes render the guest triazene significantly less basic as a consequence of the less polar nature of the CD cavity (a microsolvent effect). For any given triazene, the inhibition effect is dependent on both the size of the cyclodextrin cavity and the substituents on the cavity rims. Binding constants for 1:1 host:guest complexes increase in the order α-CD < ß-CD ~ TM-ß-CD < HP-ß-CD; in the case of α CD, formation of 2:1 complexes is also observed.