Biocatalytic and biomimetic studies of polyphenol oxidase
- Authors: Burton, Stephanie Gail
- Date: 1994
- Subjects: Phenol oxidase Polyphenols Oxidases
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4028 , http://hdl.handle.net/10962/d1004088
- Description: Mushroom polyphenol oxidase (EC 1.14.18.1) was investigated to determine its potential for application as a biocatalyst in the synthesis of o-quinones, in organic medium. In order to determine the kinetic properties of the biocatalyst, a system was devised which comprised an immobilised polyphenol oxidase extract, functioning in chloroform. The system was hydrated by the addition of buffer. A simple method for the consistent measurement of reaction rates in this heterogenous system was designed and used to obtain detailed enzyme kinetic data relating to optimisation of reaction conditions and substrate specificity. The aqueous content of the system was optimised using p-cresol as a substrate. A crude, immobilised extract of Agaricus bisporus was used to hydroxylate and oxidise a range of selected p-substituted phenolic substrates, yielding, as the sale products, o-quinones. These products were efficiently reduced to catechols by extracting the reaction mixtures with aqueous ascorbic acid solution. The biocatalytic system was also successfully utilised to produce L-DOPA, the drug used to treat Parkinson's disease, from L-acetyl tyrosine ethyl ester (ATEE). Michaelis-Menten kinetics were used to obtain apparent Km and V values with respect to the selected phenolic substrates, and the kinetic parameters obtained were found to correlate well with the steric requirements of the substrates and with their hydrophobicity. In the course of the investigation, a novel ¹H NMR method was used to facilitate measurement of the UV molar absorption coefficients of the o-quinones in reaction mixtures, thus avoiding the necessity to isolate these unstable, water-sensitive products. The biocatalytic system was extended to a continuous process, in which the immobilised enzyme was shown to function successfully in the chloroform medium for several hours, with high conversion rates. Modifications, involving partial purification and the addition of a surfactant, were investigated to determine their effect on the kinetic parameters. The results obtained using partially purified enzyme indicated that the removal of extraneous protein and/or melanoid material lead to a reduced capacity for conversion of sterically demanding substrates. The addition of the anionic detergent, sodium dodecyl sulphate (SOS), enhanced the ability of the biocatalyst to bind and oxidise sterically demanding substrates. These effects are attributed to changes in the polar state of groups within the protein binding pocket, which result in altered flexibility and hydrophobicity. Computer modelling of several biomimetic dinuclear copper complexes also indicated the importance of flexibility for effective biocatalysis. Novel binuclear copper (II complexes, containing a flexible biphenyl spacer and imidazole or benzimidazole donors, were prepared and analysed using NMR, UV, AA and cyclic voltammetric techniques. The complexes were also shown, in a detailed kinetic study, to mimic the catecholase activity of polyphenol oxidase by oxidising 3,5-di-tertbutylcatechol, and to catalyse the coupling of the phenolic substrate 2,4-di-tert-butylphenol. However, the complexes were apparently too flexible to react with smaller substrates. These biomimetic complexes provided valuable insights into the nature of the dinuclear copper binding site.
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- Date Issued: 1994
Studies in asymmetric synthesis
- Authors: Ravindran, Swarnam Shanthi
- Date: 1994
- Subjects: Asymmetric synthesis Chirality Organic compounds -- Synthesis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4352 , http://hdl.handle.net/10962/d1005017
- Description: The stereoselectivity of TiCI₄-catalysed Mukaiyama reactions of a camphor acetal-derived chiral silyl enol ether with a range of substituted aromatic aldehydes has been examined. The enantiomeric excess in each of the resulting ß-hydroxy ketones, determined by ¹H NMR spectroscopy using the lanthanide chiral shift reagent Pr(Etcf₃), ranged between 9 and 13%. The stereo-directing potential of the camphor acetal as a chiral auxiliary in the α-benzylation of carboxylate esters has been studied; the acids were chosen to illustrate substituent effects on asymmetric induction. The observed diastereoselectivity increased with increasing steric bulk of the ester group and α-benzylation of the tert-butylacetate derivative proceeded with 48% diastereoselectivity. It is proposed that the enolate adopts an endo-s-trans conformation in the transition state and preferential attack by the electrophile at the somewhat less hindered Si-face is supported by both the optical rotation data and computer modelling studies. Reductive cleavage and hydrolysis of one of the benzylated esters furnished known products from whose optical rotation the configuration of the major diastereomer was established. In order to improve the steric advantage of Si-facial attack, methods of increasing the steric bulk of the blocking group were explored. A novel 2,2-propylenedioxy hydroxycamphor acetal and its 3,3-propylenedioxy analogue were prepared. Selected carboxylate esters of these propylenedioxy acetals were subjected to α-benzylation and the 2,2-(propylenedioxy)-3-exo-tert-butylacetate derivative showed a diastereoselectivity of 57% during a-benzylation. Hydrolysis of the abenzylated phenylacetate analogue offered the known 2,3-diphenylpropanoic acid whose optical rotation indicated the preferred configuration at the new chiral centre to be (R), a result which is consistent with the proposed approach of the electrophile to the less hindered Re-face of theendo-s-trans enolate moiety and reflects an inversion of the configurational bias observed with 2-v exo-carboxylate analogues. Attempts to prepare the monocatechol acetal of the hydroxy camphor derivative although unsuccessful, led to the isolation of two novel dibornyl ethers whose structures were established by 1- and 2-D NMR spectroscopy. A study of novel applications of camphor-derived auxiliaries in the asymmetric synthesis of α-amino acids has been initiated. The several approaches tried led to the preparation of three novel dural glycine derivatives in good yield
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- Date Issued: 1994
Synthetic and spectroscopic studies of 1,4-benzodiazepine analogues
- Authors: Mphahlele, Malose Jack
- Date: 1994
- Subjects: Benzodiazepines Tranquilizing drugs
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4384 , http://hdl.handle.net/10962/d1005049
- Description: In this project, an extensive range of benzodiazepine analogues have been synthesised via Schmidt reaction of specially prepared flavanone, 4-quinolone and l-thioflavanone precursors; nitrogen insertion being effected by use of trimethylsilyl azide in trifluoroacetic acid. In some cases, several of the benzodiazepine analogues have also been prepared by alternative cyclisation routes. A detailed kinetic-mechanistic study of the Schmidt reaction of flavanones has been carried out using 'H NMR spectroscopy to explain the observed regiochemistry of nitrogen insertion. The reaction rates, for the formation of both amide and tetrazolo derivatives have been found to be influenced by the electronic effects of the A- and B-ring substituents. A series of benzodiazepine analogues have been shown to undergo regioselective A-ring chlorination with t-butylhypochlorite; the products being characterised by 'H NMR, IR and mass spectroscopy. The mass spectrometric fragmentation patterns of series of 2-aryl-4-quinolones, and 2-aryl-l ,4-benzodiazepinones and their tetrazolo[l ,5-dl analogues have been elucidated using a combination of low-resolution, high-resolution and metastable-peak analyses. The binding affinities of various benzodiazepine analogues for rat brain benzodiazepine receptors have been evaluated using a radioreceptor assay technique. Structure-activity relationships were investigated to establish the effects of various A-, B- and Coring substituents on binding affinity. The conformational preferences of selected systems have been studied using a combination of multi-pulse 'H NMR spectroscopy, X-ray crystallography and computer modelling techniques with a view to establishing the influence of conformation on binding affinity.
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- Date Issued: 1994
Synthetic and spectroscopic studies of indolizine derivatives
- Authors: Bode, Moira Leanne
- Date: 1994
- Subjects: Indole alkaloids -- Derivatives Spectrum analysis Chemistry, Organic DNA -- Synthesis
- Language: English
- Type: Thesis , Doctoral , PhD
- Identifier: vital:4385 , http://hdl.handle.net/10962/d1005050
- Description: The crystalline compound resulting from thermal cyclization of the Baylis-Hillman product, methyl 3-hydroxy-2-methylene-3-(2-pyridyl)propanoate, has been identified as the indolizine derivative, methyl indolizine-2-carboxylate, and this approach involving the reaction of pyridine-2-carboxaldehydes and acrylate analogues has been established as a general route to 2-substituted indolizines. The ease of cyclization the Baylis-Hillman products to indolizines has been shown to increase by converting the hydroxy group to an acetoxy group, and a range of acetylated Baylis-Hillman products were prepared and cyc1ized to the corresponding 2-substituted indolizines, generally in good overall yield. In the reaction of pyridine-2-carboxaldehyde and methyl vinyl ketone, the intermediate cyclized readily and directly to the corresponding indolizine. One- and two-dimensional ¹H and ¹³C NMR analysis of the 2-substituted indolizine products has permitted complete assignment of all ¹H and ¹³C NMR signals, as well as the measurement of all coupling constants for these compounds. A kinetic and mechanistic study has been conducted on the Baylis-Hillman reaction using ¹H NMR spectroscopy. A range of substrates has been examined and the reaction has been found to be third-order overall. A mechanism involving an addition - elimination sequence is proposed, which fits the kinetic data and accounts for observed substituent effects. Reaction of N,N-dimethylacrylamide with pyridine-2-carboxaldehyde in the presence of the tertiary amine catalyst, DABCO, in chloroform, yielded an unexpected product which has been identified by single crystal X-ray diffraction analysis as 1-(2,2,2-trichloro-1-hydroxyethyl)pyridine. Attempted extension of the general indolizine route to the preparation of chromene systems by reacting salicylaldehyde with methyl acrylate in the presence of DABCO, also led to an unexpected, crystalline material, identified by single crystal X-ray diffraction analysis as the coumarin derivative, 3-[(2-formylphenoxy)methyl]coumarin.A series of chloroquine analogues have been prepared from indolizine-2-carboxylic acid, pyrrolo[I,2-a]quinoline-2-carboxylic acid and imidazo[I,2-a]pyridine-2-carboxylic acid by reaction with suitable amines in the presence of the coupling reagent 1, I' -carbonyldiimidazole. This route has been shown to be a vast improvement on earlier procedures and has provided access to both secondary and tertiary indolizine-2-carboxamides. A range of N,N-dialkylindolizine-2-carboxamides have been prepared by this route, and the influence of substituents on their N-CO rotational energy barriers has been determined using variable temperature ¹H and ¹³C NMR techniques. Intercalation with natural DNA by both chloroquine and the synthesized chloroquine analogues has been examined using UV spectrophotometry, and ¹H and ³¹P NMR spectroscopy. The pyrrolo[I,2-a]quinolines have been shown to be DNA intercalators with binding affinities similar to that of the known antimalarial intercalator, chloroquine. In a preliminary study the synthesis of a short oligonucleotide has been undertaken and changes have been observed in the ¹H and ³¹P NMR spectra of the oligonucleotide on addition of the intercalator, chloroquine.
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- Date Issued: 1994