17 research outputs found

    Calixarenes: molecular modelling of and potentiometric studies on cation complexes

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    Chapter 1: The three-dimensional nature of calixarenes and their applications in sensors are reviewed. Chapter 2: Molecular mechanics was used to model the geometry of a sodium calixarene complex formed with a 1,3-alternate conformation. Partial charges were assigned to the calixarene ligands by a variety of methods. These included various semi-empirical methods (AMI, PM3 and MNDO) arbitrarily assigning formal charges to the ions and carrying out semi-empirical calculations on the full ligand:complex. An investigation into the effect of placing the cation in different starting positions was undertaken in the case of this complex also. The effect of using ligand partial charges calculated with the PM3 and MNDO semi-empirical methods is commented upon. Chapters 3 and 4: A series of calixarene phosphine oxides were used to construct ion-selective electrodes, (i.e., potentiometric sensors) and were found to be selective for calcium in the presence of a range of Group 1 and Group 2 interfering ions. Estimations of the characteristics of these electrodes (the cell constant, the Nemstian Slope Factor and potentiometric selectivity coefficients) were obtained by simple but rigorous procedure, developed for a potassium-selective valinomycin ionselective electrode. The procedure involved fitting the experimental data to a mathematical model by non-linear analysis. In this method, the interfering ion concentration is kept constant and the primary ion concentration is varied by means of a series of small volume ‘spikes’, added to a single solution, with the electrodes continually in contact, and measuring the cell potential after each addition. Monte Carlo conformational searches with molecular mechanics geometry optimisation gave very clear and positive explanations for the selectivity several calixarenes for several Group 1 and Group 2 ion, while NMR spectroscopic studies supported the conclusions drawn from the modelling studies about changes in conformation upon complexation with metal ions

    Étude de l'effet de l'insertion de chaînes latérales sur la conformation et les propriétés spectroscopiques d'oligothiophènes

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    Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal

    Feature Papers in Compounds

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    This book represents a collection of contributions in the field of the synthesis and characterization of chemical compounds, natural products, chemical reactivity, and computational chemistry. Among its contents, the reader will find high-quality, peer-reviewed research and review articles that were published in the open access journal Compounds by members of the Editorial Board and the authors invited by the Editorial Office and Editor-in-Chief

    Computer-based studies on enzyme catalysis : from structure to activity

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    Theoretical simulations are becoming increasingly important for our understanding of how enzymes work. The aim of the research presented in this thesis is to contribute to this development by applying various computational methods to three enzymes of theβ-ketoadipate pathway, and to validate the models obtained by means of quantitative structure-activity relationships (QSAR). The models and the resulting QSARs provide valuable mechanistic information about the relevant (rate-limiting) steps in the reaction cycles of the enzymes studied.Two of the enzymes that have been studied in this thesis, are flavin dependent monooxygenases: para -hydroxybenzoate hydroxylase (PHBH) from Pseudomonas fluorescence , and phenol hydroxylase (PH) from Trichosporon cutaneum . These enzymes catalyse the ortho-hydroxylation of para -hydroxybenzoate and phenol, leading to the formation of catechol and protocatechuate respectively. These products are the key intermediates in the degradation of many aromatic compounds. Once the catechol or protocatechuate is formed, the aromatic ring is cleaved between the two hydroxyl-substituted carbon atoms. This intradiol cleavage is catalysed by another enzyme studied in this thesis, catechol-1,2-dioxygenase (1,2-CTD), and by protocatechuate-3,4-dioxygenase (3,4-PCD), respectively.Catechol dioxygenaseThe reaction mechanism of catechol-1,2-dioxygenase from Pseudomonas putida has been studied by means of a QSAR approach based on gas-phase molecular orbital calculations. Catechol-1,2-dioxygenase catalyses intradiol cleavage of the aromatic ring of catechol by incorporating both oxygen atoms of molecular oxygen. In addition to the native catechol, this enzyme converts several C4-substituted catechol derivatives. In this study, the 4-methyl-, 4-fluoro-, 4-chloro-, 4-bromo, 4,5-difluoro- and 4-chloro,5-fluoro-catechols were obtained biosynthetically from the corresponding phenols by using the enzyme phenol hydroxylase. The overall rate constant for their conversion by catechol-1,2-dioxygenase was determined through steady-state kinetic experiments at various oxygen concentrations and saturating catechol concentrations.The crucial step in the reaction mechanism of the enzyme catalysed reaction was considered to be the nucleophilic attack of the substrate on the oxygen molecule. Therefore, the experimental results were compared to calculated energies of the highest occupied molecular orbital (HOMO) of the various catechol substrates, which represent their nucleophilic reactivities. A (linear) correlation was found between the calculated HOMO energies and the logarithm of the experimental rate constants. This indicates that the rate-limiting step in the overall reaction cycle involves a nucleophilic reaction of the substrate. Thus, the reaction of the substrate with molecular oxygen may indeed be rate limiting. Additional calculations excluded two other steps in the reaction cycle as being rate limiting.The results for catechol-1,2-dioxygenase from Pseudomonas putida were also compared to the data from two different types of catechol-1,2-dioxygenase, a normal (type I) and a chloro-catechol dioxygenase (type II), from Pseudomonas sp. B13. It could be argued that the difference in substrate preference between both types of catechol dioxygenases is related to a differential effect of the substituents on the rate of oxygen affinity binding by the two enzymes, rather than on the rate-limiting step.p-Hydroxybenzoate hydroxylaseAn important step that has been made in this thesis is the use of a combined quantum mechanical/molecular mechanical (QM/MM) method. Using this method, the quantum mechanical (reaction pathway) calculation of the reacting compounds could be performed within the actual environment of the protein. The surrounding protein atoms are calculated at a molecular mechanical (MM) level and their electrostatic and steric effects on the quantum mechanical system are included. This QM/MM technique has been applied to the hydroxylation step catalysed by p -hydroxybenzoate hydroxylase (PHBH). It was first investigated whether the energy barriers obtained from QM/MM reaction pathway calculations could be used to explain the variation in the overall rate constants for the conversion of a series of fluorinated substrates by PHBH. Reaction pathways were calculated for the proposed rate-limiting step in the reaction cycle: the electrophilic attack of the C4a-hydroxyperoxyflavin cofactor intermediate on the substrate. The energy profiles calculated for this reaction step with the various substrates yielded barriers with different heights. A correlation was found between the natural logarithm of the experimental overall rate constants for conversion of the fluorinated substrates by PHBH and the QM/MM calculated energy barriers for the different substrates. This correlation with overall rate constants supports that the electrophilic attack of the C4a-hydroxyperoxyflavin on the substrate is indeed the rate-limiting step in the reaction cycle.The correlation also indicates that the QM/MM model provides a realistic description of the hydroxylation step, as it accounts correctly for the effect of substrate substituents on the rate of hydroxylation. This was the basis for a further and more detailed analysis of the QM/MM model, which provided detailed insight into the mechanism of substrate and cofactor activation to facilitate the electrophilic reaction. Deprotonation of the substrate, which has been observed experimentally, is shown to significantly lower the energy barrier for the calculated reaction pathway. Also, the QM/MM model allowed the analysis of the energetic effect of the individual amino acid residues on the hydroxylation reaction. The results suggest catalytic effects of a backbone carbonyl moiety (Pro293), by a specific stabilisation of the transition state, and of a (crystal) water molecule (Wat717), which stabilises the negative charge arising on the proximal oxygen of the flavin cofactor.Phenol hydroxylaseThe QM/MM technique has also been applied to phenol hydroxylase (PH). As for PHBH, the hydroxylation step, proposed to be rate limiting in the reaction cycle of PH, has been simulated for a series of halogenated substrate derivatives. The energy barriers obtained correlate well with the logarithm of the overall rate constants. This correlation supports that the electrophilic attack of the C4a-hydroperoxyflavin on the substrate is the rate-limiting step in the reaction cycle at pH 7.6 and 25°C. An additional mechanistic question addressed in this study is the protonation state of the substrate during hydroxylation. Substrate deprotonation as a mechanism of activation has not been established for PH as firmly as it has been for PHBH. Proton transfer from phenol to a potential active site base, Asp54, has been investigated by calculating a 2-dimensional potential energy surface for the two reaction coordinates, i.e. hydroxylation and proton transfer. This potential energy surface suggests that proton transfer prior to hydroxylation is the most favourable mechanism, which indicates that in the PH reaction substrate deprotonation is important as well. The QM/MM model was further analysed to provide insight into the effect of the protein environment on the simulated reaction steps. Some catalytic effects on the hydroxylation step, i.e. of a proline carbonyl moiety and of a crystal water in the active site of PH, were similar to those found for PHBH.All together, the research presented in this thesis has made a new contribution to the development and validation of computational models that can be used to address a major challenge in the present field of biochemistry, i.e. to obtain insight into enzymatic reaction mechanisms and enzyme activity on the basis of the structure of enzyme and substrate(s). Special emphasis has been on the application and validation of the QM/MM technique in the context of a QSAR approach. The investigations of this thesis provide a first survey of the possibilities of the QM/MM method with respect to the prediction of biochemical activity, taking explicitly into account the influence of the active site surroundings.</p

    Directed evolution of phosphotriesterase: towards the efficient detoxification of sarin and soman

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    Directed evolution studies were done with PTE for the enhancement of hydrolysis of both sarin and soman analogs. Particular attention was focused on the toxic SpRc and SpSc isomers of the soman analog, and the Sp isomer of the sarin analog. Double substitution libraries yielded several mutants that had enhanced activity for the substrates. Among them was the double mutant, H257Y-L303T, which displayed a 462-fold increase in activity for hydrolysis of the most toxic SpSc isomer of the GD analog in comparison to the wild type. Several other mutants such as the triple mutants H254R-H257A-L303T and H254R-H257S-L303T had enhancements of between 150- and 200-fold, and had also displayed a different order of stereoselectivity relative to the wild type. For these mutants, the order of preferential hydrolysis was such that the SpRc isomer was preferentially hydrolyzed first. In contrast, the order of preferential hydrolysis for the wild type was that the RpRc was hydrolyzed first, followed by the RpSc, SpRc, and then the SpSc isomer. The reversal of stereoselective preference was also seen with the double substitution library members for hydrolysis of the sarin analog isomers. However, there were no significant improvements for sarin analog hydrolysis in these libraries. Among the best mutants obtained were H254G-H257W, H254G-H257R, and H257Y, all of which had catalytic efficiencies on the order of 106 M-1 s-1 for hydrolysis of the Sp isomer. The toxicity for analogs of sarin, soman, and VX was evaluated using Hydra attenuata as a model organism. The toxicity of each compound was assessed quantitatively by measuring the minimal effective concentration within 92 h in H. attenuata. There was a positive correlation between the molecular hydrophobicity of the compound and its ability to cause toxicity. Results from this study indicate the potential for application of this assay in the field of organophosphate nerve agent detection, as well as for the prediction of toxicity of structurally similar organophosphate compounds. The minimal effective concentration for two of the VX analogs was 2 orders of magnitude more toxic than the analog for soman and four orders of magnitude more toxic than the analog for sarin

    Synthesis of new microporous solids by template design

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    The rational design and synthesis of organic templates for the synthesis of new microporous materials has resulted in the production of five new materials, three of which are known to be microporous, the other two being better described as being open framework materials. The first three microporous materials are named STA-1, -2 and -3 (for St. Andrews) and have structural compositions of the form MgxAl1-xPO4 x/n Rn+.yH2O (Mg0.18Al0.82PO4.R0.094-0.22H2O, as determined by EDX for STA-1). The fourth novel microporous solid is a boron aluminophosphate (BAlPO) and the fifth an aluminophosphate solid containing its organic template intact. All of the new materials were synthesised following a systematic study over a wide range of templates including 5 homologous series designed specifically to be used as templates in the production of alumino-silicates and -phosphates. The structure of the first two magnesium aluminophosphates (STA-1, STA-2) to be solved by single microcrystal diffraction at the ESRF at Grenoble on crystals approximately 30 x 30 x 30 mum in size is described. The in situ location of the template was determined directly from x-ray diffraction in the second of these materials and has formed part of a combined experimental and computational study into template location within novel frameworks. The study shows how the encapsulation of the template is directly responsible for the similarity in pore architecture between MAPO-56 and STA- 2 (which co-crystallise) since the template adopts the same orientation in both materials Polymeric / oligomeric templates have also formed part of this study. These templates are shown to influence the phase, crystallinity, particle size and catalytic performance of the product magnesio-aluminophosphate MAPO-31. Computer modelling has also been utilised to interpret the experimental data obtained from the systematic study on the use of polymeric templates. The in situ synthesis and subsequent encapsulation of two coordination complexes inside the supercage of zeolite-Y, including the 3-methyl-1,3,5,8,12- pentaazacyclotetradecane macrocycle is also described

    Selectivity and enantioselectivity in the palladium catalysed hydrogenation of pyrazine and some substituted pyrazines

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    The palladium catalysed hydrogenation of pyrazine and some substituted pyrazines has been investigated over a range of reaction conditions.This thesis reports and discusses work in three areas. The first being the hydrogenation of pyrazine over Pd/C, second the hydrogenation of some monosubstituted pyrazines also over Pd/C and finally, the enantioselective hydrogenation of methyl pyrazine-2- carboxylate over different chirally modified catalysts.The hydrogenation of pyrazine over Pd/C and some other platinum group metal (PGM) catalysts has given an insight into its reactivity and selectivity. The interaction of pyrazine with the catalyst surface has been examined by use of deuterium-exchange which indicates pyrazine adsorbs in a co-planar manner to the surface. This has been verified experimentally by interpreting results obtained from the rates of hydrogenation for a range of aromatic N-heterocycles.The hydrogenation of methyl pyrazine-2-carboxylate and 2-pyrazinecarbonitrile (2- PCN) over Pd/C has given an insight into the effects of substituents on the reactivity of the aromatic heterocyclic ring. In the case of methyl pyrazine-2-carboxylate, hydrogenation occurs in two distinct stages. The first stage is the rapid uptake of two moles hydrogen, followed by no further hydrogenation. Investigation of the compound formed after this two-mole uptake has shown 1, 4, 5, 6-tetrahydro methyl pyrazine-2- carboxylate as the product. On an electronic level, this compound is very stable and resists further reaction due to its conjugation and is in fact an a, p-unsaturated ester having a carbamate-like structure.The unmodified hydrogenation of 2-PCN gave different results. It became evident that this compound, like methyl pyrazine-2-carboxylate before, is only partially hydrogenated because reaction stops after a two-mole hydrogen uptake. Further investigation showed the cyano substituent is being partially hydrogenated as well as the ring. However, the degree of ring/substituent hydrogenation is affected by the pH ot the reaction solvent, and the mechanism of hydrogenation is not complying with traditional observations.The major aim of this project i.e. achieving the enantioselective hydrogenation of methyl pyrazine-2-carboxylate, has been fulfilled. An enantioselective outcome has been achieved over several differently modified catalysts. This is of particular importance to the industrial collaborator in this project, Zeneca Specialities.The action of an adsorbed chiral modifier onto Pd/C is to induce the preferential formation of methyl piperazine-2-carboxylate in a typical enantiomeric excess of 2- 25%; the highest value being obtained using a cinchonidine-modified catalyst.The presented results indicate the first ever example of direct enantioselective hydrogenation of an aromatic N-heterocycle.Mechanisms for the reaction predicting the experimental observations are given

    Assessment of Molecular Modeling & Simulation

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    Electrochemical studies of gold bioaccumulation by yeast cell wall components

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    Gold, amongst other group 11 metals, was almost certainly one of the first three metals known to man. In addition to the economic importance of the metal, gold has a wide variety of applications in the medical, electrocatalytical and micro-electronics fields. However, the determination of gold ions in solution, with accuracy, precision, sensitivity and selectivity is still an interesting and much debated topic in analytical chemistry. A system whereby gold ions have been successfully detected employing an electrochemical technique, known as stripping voltammetry, has been developed. The electrochemical method was chosen over other available techniques for the sensitivity, particularly at low concentrations, and selectivity properties; notably in the presence of other metal ions. Under acidic conditions, the electrochemical technique was applied and the presence of gold(III), at a concentration of 2.53 x 10⁻⁵ mol dm⁻³ in a mine waste water sample, was detected. Biomass, in particular yeast and algal types, have been successfully employed in extracting low concentrations of gold ions from industrial effluents. The manipulation of the biological facility for mineral interaction, biohydrometallurgy, may yield numerous potential new technologies. South Africa in particular would benefit from this area of research, since the country is a major ore and metal refining country and if the output and the efficiency of the mines could be improved, even by a small percentage, the financial rewards would be vast. In this study, the application of adsorptive cathodic stripping voltammetry (AdCSV) of gold(III) in the presence of various Saccharomyces cerevisiae cell wall components, was investigated to determine which, if any, were involved specifically in the chemical binding of the gold ions. The chitin and mannan extracts showed the most promise with detection limits of 1.10 x 10⁻⁶ mol dm⁻³ and 9 x 10⁻⁹ mol dm⁻³, respectively; employing the AdCSV technique. A modification of the stripping voltammetry technique, Osteryoung square wave stripping voltammetry (OSWSV), provided the lowest detection limit, for gold(IIl) in the presence of mannan, of 1.70 x 10⁻¹¹ mol dm⁻³ ; utilising a modified carbon paste electrode. The detection of gold(III) has been shown to be dependent on the type of electrode employed, the electrolyte solution and the presence of interfering agents. The effect of copper(II) and silver(I) on the detection of the gold(III) in solution was investigated; whilst the silver(I) has shown no detrimental effects on gold (III) detection systems, copper(II) has indicated the possibility of forming an inter-metallic compound with the gold(III). However, mannan has shown to selectively and preferentially bind the gold(III) in the presence of a ten-fold excess of copper(II). Nuclear magnetic resonance (NMR) and infrared (IR) spectroscopy, as well as computer modelling techniques were employed to further investIgate the mannan-gold(III) interaction and proposed complex formed. The NMR, IR and computer modelling data are in agreement with the electrochemical data on proposing a mannan-gold(III) complex. The co-ordination site was established to be in the vicinity of the H-I and H-2 protons and the gold(III) adopts a square-planar geometry upon co-ordination. The benefits of the research are useful from a biological perspective (i. e. as more is known about the binding sites, microbiologists/biochemists may work on the optimisation of parameters for these sites or work could be furthered into the enhanced expression of the sites) and an industrial one. In addition to the' two major benefits, an improved understanding of gold and its chemistry would be achieved, which is advantageous for other fields of research as well
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