Radical chemistry of the catechol/quinone system and its role in the control of lipid peroxidation and melanin biosynthesis

The catechol (1,2-dihydroxybenzene) is a privileged structural motif among natural antioxidants like flavonoids, owing to its reactivity with alkylperoxyl radicals due to the stability of the semiquinone radical. The exploration of the relevance and mechanism of this non-conventional antioxidant chemistry in heterogenous biomimetic systems (aqueous micelles and unilamellar liposomes) is explored for the first time in Chapter 1. Results show antioxidant behaviour that surpasses that of nature’s premiere antioxidant α-tocopherol and relies on the cross-dismutation of alkylperoxyl and hydroperoxyl radicals at the water-lipid interface with regeneration of the catechol function from the oxidized quinone. The design and synthesis of new biomimetic catechol-type antioxidants by conjugation of thiols (e.g. cysteine) with quinones highlighted an unusual 1,6-type regioselectivity, which had been previously reported but never fully rationalized. Owing to its importance both in nature and in the development of new antioxidants, we investigated it in detail in Chapter 2. We could prove the onsetting of a radical-chain mechanism intermediated by thiyl and thiosemiquinone radicals at the basis of the “anomalous nucleophilic addition” of thiols to ortho-quinones, which paves the way to better understanding of the chemistry of such systems. The oxidation of catechols to the corresponding quinones is also a key reaction in the biosynthesis of melanins, mediated by enzyme Tyrosinase.

Catalytic liquid- and gas-phase oxidations for the synthesis of intermediates and specialty chemicals: some examples of industrial relevance

Nowadays, it is clear that the target of creating a sustainable future for the next generations requires to re-think the industrial application of chemistry. It is also evident that more sustainable chemical processes may be economically convenient, in comparison with the conventional ones, because fewer by-products means lower costs for raw materials, for separation and for disposal treatments; but also it implies an increase of productivity and, as a consequence, smaller reactors can be used. In addition, an indirect gain could derive from the better public image of the company, marketing sustainable products or processes. In this context, oxidation reactions play a major role, being the tool for the production of huge quantities of chemical intermediates and specialties. Potentially, the impact of these productions on the environment could have been much worse than it is, if a continuous efforts hadn’t been spent to improve the technologies employed. Substantial technological innovations have driven the development of new catalytic systems, the improvement of reactions and process technologies, contributing to move the chemical industry in the direction of a more sustainable and ecological approach. The roadmap for the application of these concepts includes new synthetic strategies, alternative reactants, catalysts heterogenisation and innovative reactor configurations and process design. Actually, in order to implement all these ideas into real projects, the development of more efficient reactions is one primary target. Yield, selectivity and space-time yield are the right metrics for evaluating the reaction efficiency. In the case of catalytic selective oxidation, the control of selectivity has always been the principal issue, because the formation of total oxidation products (carbon oxides) is thermodynamically more favoured than the formation of the desired, partially oxidized compound. As a matter of fact, only in few oxidation reactions a total, or close to total, conversion is achieved, and usually the selectivity is limited by the formation of by-products or co-products, that often implies unfavourable process economics; moreover, sometimes the cost of the oxidant further penalizes the process. During my PhD work, I have investigated four reactions that are emblematic of the new approaches used in the chemical industry. In the Part A of my thesis, a new process aimed at a more sustainable production of menadione (vitamin K3) is described. The “greener” approach includes the use of hydrogen peroxide in place of chromate (from a stoichiometric oxidation to a catalytic oxidation), also avoiding the production of dangerous waste. Moreover, I have studied the possibility of using an heterogeneous catalytic system, able to efficiently activate hydrogen peroxide. Indeed, the overall process would be carried out in two different steps: the first is the methylation of 1-naphthol with methanol to yield 2-methyl-1-naphthol, the second one is the oxidation of the latter compound to menadione. The catalyst for this latter step, the reaction object of my investigation, consists of Nb2O5-SiO2 prepared with the sol-gel technique. The catalytic tests were first carried out under conditions that simulate the in-situ generation of hydrogen peroxide, that means using a low concentration of the oxidant. Then, experiments were carried out using higher hydrogen peroxide concentration. The study of the reaction mechanism was fundamental to get indications about the best operative conditions, and improve the selectivity to menadione. In the Part B, I explored the direct oxidation of benzene to phenol with hydrogen peroxide. The industrial process for phenol is the oxidation of cumene with oxygen, that also co-produces acetone. This can be considered a case of how economics could drive the sustainability issue; in fact, the new process allowing to obtain directly phenol, besides avoiding the co-production of acetone (a burden for phenol, because the market requirements for the two products are quite different), might be economically convenient with respect to the conventional process, if a high selectivity to phenol were obtained. Titanium silicalite-1 (TS-1) is the catalyst chosen for this reaction. Comparing the reactivity results obtained with some TS-1 samples having different chemical-physical properties, and analyzing in detail the effect of the more important reaction parameters, we could formulate some hypothesis concerning the reaction network and mechanism. Part C of my thesis deals with the hydroxylation of phenol to hydroquinone and catechol. This reaction is already industrially applied but, for economical reason, an improvement of the selectivity to the para di-hydroxilated compound and a decrease of the selectivity to the ortho isomer would be desirable. Also in this case, the catalyst used was the TS-1. The aim of my research was to find out a method to control the selectivity ratio between the two isomers, and finally to make the industrial process more flexible, in order to adapt the process performance in function of fluctuations of the market requirements. The reaction was carried out in both a batch stirred reactor and in a re-circulating fixed-bed reactor. In the first system, the effect of various reaction parameters on catalytic behaviour was investigated: type of solvent or co-solvent, and particle size. With the second reactor type, I investigated the possibility to use a continuous system, and the catalyst shaped in extrudates (instead of powder), in order to avoid the catalyst filtration step. Finally, part D deals with the study of a new process for the valorisation of glycerol, by means of transformation into valuable chemicals. This molecule is nowadays produced in big amount, being a co-product in biodiesel synthesis; therefore, it is considered a raw material from renewable resources (a bio-platform molecule). Initially, we tested the oxidation of glycerol in the liquid-phase, with hydrogen peroxide and TS-1. However, results achieved were not satisfactory. Then we investigated the gas-phase transformation of glycerol into acrylic acid, with the intermediate formation of acrolein; the latter can be obtained by dehydration of glycerol, and then can be oxidized into acrylic acid. Actually, the oxidation step from acrolein to acrylic acid is already optimized at an industrial level; therefore, we decided to investigate in depth the first step of the process. I studied the reactivity of heterogeneous acid catalysts based on sulphated zirconia. Tests were carried out both in aerobic and anaerobic conditions, in order to investigate the effect of oxygen on the catalyst deactivation rate (one main problem usually met in glycerol dehydration). Finally, I studied the reactivity of bifunctional systems, made of Keggin-type polyoxometalates, either alone or supported over sulphated zirconia, in this way combining the acid functionality (necessary for the dehydrative step) with the redox one (necessary for the oxidative step). In conclusion, during my PhD work I investigated reactions that apply the “green chemistry” rules and strategies; in particular, I studied new greener approaches for the synthesis of chemicals (Part A and Part B), the optimisation of reaction parameters to make the oxidation process more flexible (Part C), and the use of a bioplatform molecule for the synthesis of a chemical intermediate (Part D).

Neuroprotective strategies against neurodegeneration in cellular model systems

In the present study we analyzed new neuroprotective therapeutical strategies in PD (Parkinson’s disease) and AD (Alzheimer’s disease). Current therapeutic strategies for treating PD and AD offer mainly transient symptomatic relief but it is still impossible to block the loss of neuron and then the progression of PD and AD. There is considerable consensus that the increased production and/or aggregation of α- synuclein (α-syn) and β-amyloid peptide (Aβ), plays a central role in the pathogenesis of PD, related synucleinopathies and AD. Therefore, we identified antiamyloidogenic compounds and we tested their effect as neuroprotective drug-like molecules against α-syn and β-amyloid cytotoxicity in PC12. Herein, we show that two nitro-catechol compounds (entacapone and tolcapone) and 5 cathecol-containing compounds (dopamine, pyrogallol, gallic acid, caffeic acid and quercetin) with antioxidant and anti-inflammatory properties, are potent inhibitors of α-syn and β-amyloid oligomerization and fibrillization. Subsequently, we show that the inhibition of α-syn and β-amyloid oligomerization and fibrillization is correlated with the neuroprotection of these compounds against the α-syn and β-amyloid-induced cytotoxicity in PC12. Finally, we focused on the study of the neuroprotective role of microglia and on the possibility that the neuroprotection properties of these cells could be use as therapeutical strategy in PD and AD. Here, we have used an in vitro model to demonstrate neuroprotection of a 48 h-microglial conditioned medium (MCM) towards cerebellar granule neurons (CGNs) challenged with the neurotoxin 6-hydroxydopamine (6-OHDA), which induces a Parkinson-like neurodegeneration, with Aβ42, which induces a Alzheimer-like neurodegeneration, and glutamate, involved in the major neurodegenerative diseases. We show that MCM nearly completely protects CGNs from 6-OHDA neurotoxicity, partially from glutamate excitotoxicity but not from Aβ42 toxin.

Changes in plant metabolism induced by dioxygenase inhibitors and their effect on the epiphytic microbial community and fire blight (Erwinia amylovora) control

Fire blight, caused by the gram negative bacterium Erwinia amylovora, is one of the most destructive bacterial diseases of Pomaceous plants. Therefore, the development of reliable methods to control this disease is desperately needed. This research investigated the possibility to interfere, by altering plant metabolism, on the interactions occurring between Erwinia amylovora, the host plant and the epiphytic microbial community in order to obtain a more effective control of fire blight. Prohexadione-calcium and trinexapac-ethyl, two dioxygenase inhibitors, were chosen as a chemical tool to influence plant metabolism. These compounds inhibit the 2-oxoglutarate-dependent dioxygenases and, therefore, they greatly influence plant metabolism. Moreover, dioxygenase inhibitors were found to enhance plant resistance to a wide range of pathogens. In particular, dioxygenase inhibitors application seems a promising method to control fire blight. From cited literature, it is assumed that these compounds increase plant defence mainly by a transient alteration of flavonoids metabolism. We tried to demonstrate, that the reduction of susceptibility to disease could be partially due to an indirect influence on the microbial community established on plant surface. The possibility to influence the interactions occurring in the epiphytic microbial community is particularly interesting, in fact, the relationships among different bacterial populations on plant surface is a key factor for a more effective biological control of plant diseases. Furthermore, we evaluated the possibility to combine the application of dioxygenase inhibitors with biological control in order to develop an integrate strategy for control of fire blight. The first step for this study was the isolation of a pathogenic strain of E. amylovora. In addition, we isolated different epiphytic bacteria, which respond to general requirements for biological control agents. Successively, the effect of dioxygenase inhibitors treatment on microbial community was investigated on different plant organs (stigmas, nectaries and leaves). An increase in epiphytic microbial population was found. Further experiments were performed with aim to explain this effect. In particular, changes in sugar content of nectar were observed. These changes, decreasing the osmotic potential of nectar, might allow a more consistent growth of epiphytic bacteria on blossoms. On leaves were found similar differences as well. As far as the interactions between E. amylovora and host plant, they were deeply investigated by advanced microscopical analysis. The influence of dioxygenase inhibitors and SAR inducers application on the infection process and migration of pathogen inside different plant tissues was studied. These microscopical techniques, combined with the use of gpf-labelled E. amylovora, allowed the development of a bioassay method for resistance inducers efficacy screening. The final part of the work demonstrated that the reduction of disease susceptibility observed in plants treated with prohexadione-calcium is mainly due to the accumulation of a novel phytoalexins: luteoforol. This 3-deoxyflavonoid was proven to have a strong antimicrobial activity.

Oxylipins biosynthesis in Lactobacillus helveticus in the presence of oxidative stress

This PhD thesis is aimed at studying the possible pathways and the mechanisms that can trigger oxylipins biosynthesis, and particularly that of short chain aldehydes and alcohols, in Lactobacillus helveticus, also in the presence of oxidative stress, using a totally labelled linoleic acid as precursor. In plants and fungi these molecules, involved in defence mechanisms against pathogens and in communication systems, derive from the oxidation of cellular unsaturated fatty acids (UFAs) and their accumulation is associated with stress exposure. Since some oxylipins are produced also by lactobacilli, it is possible to hypothesize that a metabolic pathway from UFAs to oxylipins, similar to what happens in plants and fungi, is present also in lactic acid bacteria. The results obtained pointed out that some volatile molecules are the result of UFAs catabolism, since they appear only when cells are incubated in their presence. Labelled linoleic acid is integrated in the membrane and subsequently transformed into aldehydes and alcohols, whose extent and carbon atoms number depend on stress exposure. The enzymes responsible for this metabolic pathway in plants and fungi (e.g. lipoxygenase, dioxygenase) seem to be absent in Lactobacillus helveticus and in other lactobacilli. Proteomic analyses show the over expression of many proteins, including thioredoxin reductase (part of the bacterial oxidative defence system), mainly in cells grown with linoleic acid without oxidative stress exposure, confirming that linoleic acid itself induces oxidative stress. 6 general oxidoreductases (class including dioxygenases and peroxidase) were found and therefore a deeper investigation on them could be productive in elucidating all steps involved in oxylipins biosynthesis in bacteria. Due to the multiple role of oxylipins (flavouring agents, antimicrobial compounds and interspecific signalling molecules) the identification of genes involved and regulating factors should have an important biotechnological impact, also allowing the overproduction of selected bioactive molecules.

Fully bio-based epoxy resins

Epoxy resins are mainly produced by reacting bisphenol A with epichlorohydrin. Growing concerns about the negative health effects of bisphenol A are urging researchers to find alternatives. In this work diphenolic acid is suggested, as it derives from levulinic acid, obtained from renewable resources. Nevertheless, it is also synthesized from phenol, from fossil resources, which, in the current paper has been substituted by plant-based phenols. Two interesting derivatives were identified: diphenolic acid from catechol and from resorcinol. Epichlorohydrin on the other hand, is highly carcinogenic and volatile, leading to a tremendous risk of exposure. Thus, two approaches have been investigated and compared with epichlorohydrin. The resulting resins have been characterized to find an appropriate application, as epoxy are commonly used for a wide range of products, ranging from composite materials for boats to films for food cans. Self-curing capacity was observed for the resin deriving from diphenolic acid from catechol. The glycidyl ether of the diphenolic acid from resorcinol, a fully renewable compound, was cured in isothermal and non-isothermal tests tracked by DSC. Two aliphatic amines were used, namely 1,4-butanediamine and 1,6-hexamethylendiamine, in order to determine the effect of chain length on the curing of an epoxy-amine system and determine the kinetic parameters. The latter are crucial to plan any industrial application. Both diamines demonstrated superior properties compared to traditional bisphenol A-amine systems.

New and More Sustainable Processes for the Synthesis of Phenolics: 2-phenoxyethanol and hydroxytyrosol

The research work has dealt with the study of new catalytic processes for the synthesis of fine chemicals belonging to the class of phenolics, namely 2-phenoxyethanol and hydroxytyrosol. The two synthetic procedures investigated have the advantages of being much closer to the Green Chemistry principles than those currently used industrially. In both cases, the challenge was that of finding catalysts and methods which led to the production of less waste, and used less hazardous chemicals, safer solvents, and reusable heterogeneous catalysts. In the case of 2-phenoxyethanol, the process investigated involves the use of ethylene carbonate (EC) as the reactant for phenol O-hydroxyethylation, in place of ethylene oxide. Besides being a safer reactant, the major advantage of using EC in the new synthesis is the better selectivity to the desired product achieved. Moreover, the solid catalyst based on Na-mordenite was fully recyclable. The reaction mechanism and the effect of the Si/Al ratio in the mordenite were investigated. In the case of hydroxytyrosol, which is one of the most powerful natural antioxidants, a new synthetic procedure was investigated; in fact, the method currently employed, the hydrolysis of oleuropein, an ester extracted from the waste water processing of the olive, makes use of large amounts of organic solvents (hexane, ethyl acetate), and involves several expensive steps of purification. The synthesis procedure set up involves first the reaction between catechol and 2,2-dimethoxyacetaldehyde, followed by the one-pot reduction of the intermediate to give the desired product. Both steps were optimized, in terms of catalyst used, and of reaction conditions, that allowed to reach ca 70% yield in each step. The reaction mechanism was investigated and elucidated. During a 3-month period spent at the University of Valencia (with Prof. A. Corma’s group), a process for the production of diesel additives (2,5-bis(propoxymethyl)furan) from fructose has been investigated.

Hierarchically organized chitin-based matrices

Chitin is the second most abundant biopolymer on Earth and the most diffused across the known species, being present in more than 70 % of them. It is present in a huge variety of different structures and morphologies being a massive pool of information for new material science approach. This thesis aims to study chitin at different level of organization using diverse approaches. Three main topics are discussed in this manuscript. The first is the use of a bottom-up approach to study chitin nanofibrils self-assembly in water triggering the assembly by pH increment. Successively, the assembly was studied in presence of another pH responsive biomacromolecule, the collagen, to get new composite materials and study how the assembly and the chitin/collagen ratio influence fibroblast’s viability. The second topic focuses on biogenic organized chitin-based matrices, in both Ariolimax californicus and Loligo vulgaris. This study aims to understand the features that give raise to the properties of those matrices. Finally, in the last section a top-up approach was used to exploit natural hierarchically organized chitinous matrices to obtain organized functional materials introducing a catechol on the free amino group of deacetylated chitin (DA 77 %). In conclusion, this study of chitin at different level of organization emphasized different novelties depending on the organization level studied. Moreover, this thesis gives many possibilities for future bioinspired routes to get highly organized materials, or for highly organized functional materials based on natural chitin-based matrices.

Design and synthesis of (pro)electrophilic compounds for investigating the multifactorial nature of neurodegenerative diseases: focus on inflammation-driven events

Neuroinflammation constitutes a major player in the etiopathology of neurodegenerative diseases (NDDs), by orchestrating several neurotoxic pathways which in concert lead to neurodegeneration. A positive feedback loop occurs between inflammation, microglia activation and misfolding processes that, alongside excitotoxicity and oxidative events, represent crucial features of this intricate scenario. The multi-layered nature of NDDs requires a deepen investigation on how these vicious cycles work. This could further help in the search for effective treatments. Electrophiles are critically involved in the modulation of a variety of neuroprotective responses. Thus, we envisioned their peculiar ability to switch on/off biological activities as a powerful tool for investigating the neurotoxic scenario driven by inflammation in NDDs. In particular, in this thesis project, we wanted to dissect at a molecular level the functional role of (pro)electrophilic moieties of previously synthesized thioesters of variously substituted trans-cinnamic acids, to identify crucial features which could interfere with amyloid aggregation as well as modulate Nrf2 and/or NF-κB activation. To this aim, we first synthesized new compounds to identify bioactive cores which could specifically modulate the intended target. Then, we systematically modified their structure to reach additional pathogenic pathways which could in tandem contribute to the inflammatory process. In particular, following the investigation of the mechanistic underpinnings involving the catechol feature in amyloid binding through the synthesis of new dihydroxyl derivatives, we incorporated the identified antiaggregating nucleus into constrained frames which could contrast neuroinflammation also through the modulation of CB2Rs. In parallel, Nrf2 and/or NF-κB antinflammatory structural requirements were combined with the neuroprotective cores of pioglitazone, an antidiabetic drug endowed with MAO-B inhibitory properties, and memantine, which notably contrasts excitotoxicity. By acting as Swiss army knives, the new set of molecules emerge as promising tools to deepen our insights into the complex scenario regulating NDDs.