Biocatalytic and biomimetic studies of polyphenol oxidase

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

Isolation and Identification of Bioactive Secondary Metabolites

The health benefits of food, plants, fruits, and seaweeds stem from the biological activities of their constituents—namely, secondary metabolites. The study of secondary metabolites and their potential to treat and/or prevent a number of diseases has become a research topic of growing interest for biologists, pharmacists, and chemists. Notably, in order to propose a compound as a potential new drug with pharmacological effects, the chemical structure of this compound and its biological activity against a given target must be well established. The Special Issue, “Isolation and Identification of Bioactive Secondary Metabolites”, considers species beyond their nutritional value and identifies instances of wider and more efficient use, thereby contributing to a more sustainable management of natural resources. The fifteen articles published in this Special Issue reflect the latest research trends, and consider the isolation, identification, and assessment of the beneficial effects of secondary metabolites from both edible and inedible species. Thus, these contributions collectively demonstrate that these compounds, and their plants of origin, should be valued beyond their nutritional benefits

From a multitarget antidiabetic glycosyl isoflavone towards new molecular entities against diabetes and Alzheimer’s disease : generation of lead series and target assessment

Type 2 diabetes and Alzheimer’s disease are closely related amyloid diseases globally affecting millions of people. However, the pathophysiological mechanisms connecting both diseases still require further investigation. In this work, we compile the existing evidence in the literature to allow the establishment of etiological links needed for drug discovery against diabetes-induced dementia. Furthermore, we provide an extensive revision of bioactive lead molecules that encourage further studies, particularly focusing on polyphenol sugar conjugates endowed with antidiabetic and neuroprotective activities. The state-of-the-art synthetic approaches for the generation of these types of molecules are also covered, thus setting the organic chemistry background for the original research work here developed. The use of carbohydrate-based molecules in drug research and development has multiple recognized benefits. In addition to enhanced solubility, bioavailability, and antidiabetic effects as previously reported, in this work we show, for the first time, that C-glucosylation is able to reverse the membrane dipole potential decrease induced by planar lipophilic polyphenols, elsewhere described as Pan-Assay Interference Compounds. This is a relevant discovery for drug development, particularly in the context of this thesis due to the polyphenolic nature of the compounds here presented. One of these compounds, 8-β-D-glucosylgenistein, was investigated in a diet-induced obese mouse of type 2 diabetes and found to exert a multitarget antidiabetic mechanism of action that goes beyond prior conjectures. Indeed, this antihyperglycemic glucosyl isoflavone reduces the renal threshold for glucose reabsorption, ameliorates diabetes-associated non-alcoholic fatty liver disease and hypercholesterolemia, normalizes insulin-degrading enzyme expression, and increases glucosestimulated insulin secretion. However, the detected inability of this polyphenol to permeate the blood brain barrier and to exert neuroprotective effects encouraged the pursuit of new scaffolds with therapeutic potential against diabetes-induced dementia. The role of amyloid β in the neurodegenerative processes occurring in Alzheimer’s disease and diabetes-induced dementia is, nowadays, unquestionable. Yet, targeted therapies aimed at inhibiting amyloid secretion or aggregation have, so far, failed clinical trials. In the past decade, the role of the cellular prion protein (PrPC) – a high-affinity ligand of amyloid β oligomers (Aβo) – has, in fact, been regarded as the limiting step in the cascade of events leading to neurodegeneration. Fyn kinase is one of the key players in this cascade, which culminates with the formation of neurofibrillary tangles composed by hyperphosphorylated tau, eventually leading to cell death. In this perspective, we have identified innovative N-methylpiperazinyl flavones and their glucosyl derivatives as Aβo-binders and non-toxic disruptors of Aβo-PrPC interactions. Furthermore, easily accessed glucosyl polyphenols with improved pharmacokinetic properties were also investigated and revealed to inhibit Aβ-induced Fyn activation with concomitant decrease in tau phosphorylation. Fyn kinase inhibition is considered a novel therapeutic strategy for Alzheimer’s disease, and these compounds are the first to accomplish this goal, with proven downstream effects. These molecules thus share the potential for further development against Alzheimer’s disease and diabetes-induced dementia. The work presented in this thesis elucidates the therapeutic relevance of natural and nature-inspired C-glucosyl polyphenols in the studied biological context, and highlights the usefulness of carbohydrate-based molecules for medicinal chemistry applications