Microwave-Mediated Hetero Diels-Alder reaction: Synthesis of biologically active compounds

Heterocyclic compounds represent almost two-thirds of all the known organic compounds: they are widely distributed in nature and play a key role in a huge number of biologically important molecules including some of the most significant for human beings. A powerful tool for the synthesis of such compounds is the hetero Diels-Alder reaction (HDA), that involve a [4+2] cycloaddition reaction between heterodienes and suitable dienophiles. Among heterodienes to be used in such six-membered heterocyclic construction strategy, 3-trialkylsilyloxy-2-aza-1,3-dienes (Fig 1) has been demonstrated particularly attractive. In this thesis work, HDA reactions between 2-azadienes and carbonylic and/or olefinic dienophiles, are described. Moreover, substitution of conventional heating by the corresponding dielectric heating as been explored in the frame of Microwave-Assisted-Organic-Synthesis (MAOS) which constitutes an up-to-grade research field of great interest both from an academic and industrial point of view. Reaction of the azadiene 1 (Fig 1) will be described using as dienophiles carbonyl compounds as aldehyde and ketones. The six-membered adducts thus obtained (Scheme 1) have been elaborated to biologically active compounds like 1,3-aminols which constitutes the scaffold for a wide range of drugs (Prozac®, Duloxetine, Venlafaxine) with large applications in the treatment of severe diseases of nervous central system (NCS). Scheme 1 The reaction provides the formation of three new stereogenic centres (C-2; C-5; C-6). The diastereoselective outcome of these reactions has been deeply investigated by the use of various combination of achiral and chiral azadienes and aliphatic, aromatic or heteroaromatic aldehydes. The same approach, basically, has been used in the synthesis of piperidin-2-one scaffold substituting the carbonyl dienophile with an electron poor olefin. Scheme 2 As a matter of fact, this scaffold is present in a very large number of natural substances and, more interesting, is a required scaffold for an huge variety of biologically active compounds. Activated olefins bearing one or two sulfone groups, were choose as dienophiles both for the intrinsic characteristic flexibility of the “sulfone group” which may be easily removed or elaborated to more complex decorations of the heterocyclic ring, and for the electron poor property of this dienophiles which makes the resulting HDA reaction of the type “normal electron demand”. Synthesis of natural compounds like racemic (±)-Anabasine (alkaloid of Tobacco’s leaves) and (R)- and (S)-Conhydrine (alkaloid of Conium Maculatum’s seeds and leaves) and its congeners, are described (Fig 2).

Stereoselective synthesis of heterocycles as intermediates of biologically active compounds

The aim of this thesis was to investigate the synthesis of enantiomerically enriched heterocycles and dehydro-β-amino acid derivatives which can be used as scaffolds or intermediates of biologically active compounds, in particular as novel αvβ3 and α5β1 integrin ligands. The starting materials of all the compounds here synthesized are alkylideneacetoacetates. Alkylidene derivates are very usefull compounds, they are usually used as unsaturated electrophiles and they have the advantage of introducing different kind of functionality that may be further elaborated. In chapter 1, regio- and stereoselective allylic amination of pure carbonates is presented. The reaction proceeds via uncatalyzed or palladium-catalyzed conditions and affords enantiopure dehydro-β-amino esters that are useful precursor of biologically active compounds. Chapter 2 illustrates the synthesis of substituted isoxazolidines and isoxazolines via Michael addition followed by intramolecular hemiketalisation. The investigation on the effect of the Lewis acid catalysis on the regioselectivity of the addition it also reported. Isoxazolidines and isoxazolines are interesting heterocyclic compounds that may be regarded as unusual constrained -amino acids or as furanose mimetics. The synthesis of unusual cyclic amino acids precursors, that may be envisaged as proline analogues, as scaffolds for the design of bioactive peptidomimetics is presented in chapter 3. The synthesis of 2-substituted-3,4-dehydropyrrole derivatives starting from allylic carbonates via a two step allylic amination/ring closing metathesis (RCM) protocol is carried out. The reaction was optimized by testing different Grubbs’ catalysts and carbamate nitrogen protecting groups. Moreover, in view of a future application of these dehydro-β-amino acids as central core of peptidomimetics , the malonate chain was also used to protect nitrogen prior to RCM. Finally, chapter 4 presents the synthesis of two novel different classes of integrin antagonists, one derived from dehydro-β-amino acid prepared as described in chapter 1 and the other one has isoxazolidines synthesized in chapter 2 as rigid constrained core. Since that these compounds are promising RGD mimetics for αvβ3 and α5β1 integrins, they have been submitted to biological assay. and to interpret on a molecular basis their different affinities for the αvβ3 receptor, docking studies were performed using Glide program.

Surface active substances in the water column during POLARSTERN cruise ANT-XXI/3 (EIFEX)

Surface active substances (SAS) in the water column were measured by voltammetry using the electrochemical probe o-nitrophenol (ONP) during EIFEX, a mesoscale open ocean iron enrichment experiment in the Southern Ocean. SAS levels were low throughout the experiment (<0.005 - 0.03 mg/L Triton X-100 equivalents). Initially SAS was extremely low in the photic zone, but as the phytoplankton bloom developed concentrations markedly increased throughout the upper 100 m (~0.02 mg/L Triton X-100 equivalents). Highest concentrations of SAS (>0.02 mg/L Triton X-100 equivalents) were found at the end of the bloom particularly at density discontinuities where organic material may accumulate. Exudates from diatoms appeared to be the major source of SAS during EIFEX, either from direct extracellular release or in the action of being grazed upon by zooplankton.

Surface-active phospholipid (surfactant) in equine tendon and tendon sheath fluid

AIM: To investigate the presence of surface-active phospholipid (SAPL, or surfactant) in equine tendon and tendon sheath fluid. METHODS: The left front flexor tendon and sheath were removed from five Thoroughbred horses. Phospholipid was extracted from tendon sheath fluid using Folch reagent and quantified using spectroscopy. Transmission electron microscopy (TEM) was used to observe the tendon surfaces. RESULTS: The presence of phospholipid (90.6 (SD 4.3) mu g/ml) in tendon sheath fluid, plus the appearance of oligolamellar layers and lamellar bodies on the tendon surface were indicative of SAPL. CONCLUSIONS: Evidence of SAPL was found in equine tendon, and may have a similar lubricating function as reported for synovial joints. CLINICAL RELEVANCE: These findings may have important implications for normal tendon function and possible therapeutic adjuncts for tendon and tendon sheath injuries.

Development of nanostructured hydrogel for spatial and temporal controlled release of active compounds

L’utilisation de nanovecteurs pour la livraison contrôlée de principes actifs est un concept commun de nous jours. Les systèmes de livraison actuels présentent encore cependant des limites au niveau du taux de relargage des principes actifs ainsi que de la stabilité des transporteurs. Les systèmes composés à la fois de nanovecteurs (liposomes, microgels et nanogels) et d’hydrogels peuvent cependant permettre de résoudre ces problèmes. Dans cette étude, nous avons développé un système de livraison contrôlé se basant sur l’incorporation d’un nanovecteur dans une matrice hydrogel dans le but de combler les lacunes des systèmes se basant sur un vecteur uniquement. Une telle combinaison pourrait permettre un contrôle accru du relargage par stabilisation réciproque. Plus spécifiquement, nous avons développé un hydrogel structuré intégrant des liposomes, microgels et nanogels séparément chargés en principes actifs modèles potentiellement relargués de manière contrôlé. Ce contrôle a été obtenu par la modification de différents paramètres tels que la température ainsi que la composition et la concentration en nanovecteurs. Nous avons comparé la capacité de chargement et la cinétique de relargage de la sulforhodamine B et de la rhodamine 6G en utilisant des liposomes de DOPC et DPPC à différents ratios, des nanogels de chitosan/acide hyaluronique et des microgels de N-isopropylacrylamide (NIPAM) à différents ratios d’acide méthacrylique, incorporés dans un hydrogel modèle d’acrylamide. Les liposomes présentaient des capacités de chargement modérés avec un relargage prolongé sur plus de dix jours alors que les nanogels présentaient des capacités de chargement plus élevées mais une cinétique de relargage plus rapide avec un épuisement de la cargaison en deux jours. Comparativement, les microgels relarguaient complétement leur contenu en un jour. Malgré une cinétique de relargage plus rapide, les microgels ont démontré la possibilité de contrôler finement le chargement en principe actif. Ce contrôle peut être atteint par la modification des propriétés structurelles ou en changeant le milieu d’incubation, comme l’a montré la corrélation avec les isothermes de Langmuir. Chaque système développé a démontré un potentiel contrôle du taux de relargage, ce qui en fait des candidats pour des investigations futures.

Development of nanostructured hydrogel for spatial and temporal controlled release of active compounds

L’utilisation de nanovecteurs pour la livraison contrôlée de principes actifs est un concept commun de nous jours. Les systèmes de livraison actuels présentent encore cependant des limites au niveau du taux de relargage des principes actifs ainsi que de la stabilité des transporteurs. Les systèmes composés à la fois de nanovecteurs (liposomes, microgels et nanogels) et d’hydrogels peuvent cependant permettre de résoudre ces problèmes. Dans cette étude, nous avons développé un système de livraison contrôlé se basant sur l’incorporation d’un nanovecteur dans une matrice hydrogel dans le but de combler les lacunes des systèmes se basant sur un vecteur uniquement. Une telle combinaison pourrait permettre un contrôle accru du relargage par stabilisation réciproque. Plus spécifiquement, nous avons développé un hydrogel structuré intégrant des liposomes, microgels et nanogels séparément chargés en principes actifs modèles potentiellement relargués de manière contrôlé. Ce contrôle a été obtenu par la modification de différents paramètres tels que la température ainsi que la composition et la concentration en nanovecteurs. Nous avons comparé la capacité de chargement et la cinétique de relargage de la sulforhodamine B et de la rhodamine 6G en utilisant des liposomes de DOPC et DPPC à différents ratios, des nanogels de chitosan/acide hyaluronique et des microgels de N-isopropylacrylamide (NIPAM) à différents ratios d’acide méthacrylique, incorporés dans un hydrogel modèle d’acrylamide. Les liposomes présentaient des capacités de chargement modérés avec un relargage prolongé sur plus de dix jours alors que les nanogels présentaient des capacités de chargement plus élevées mais une cinétique de relargage plus rapide avec un épuisement de la cargaison en deux jours. Comparativement, les microgels relarguaient complétement leur contenu en un jour. Malgré une cinétique de relargage plus rapide, les microgels ont démontré la possibilité de contrôler finement le chargement en principe actif. Ce contrôle peut être atteint par la modification des propriétés structurelles ou en changeant le milieu d’incubation, comme l’a montré la corrélation avec les isothermes de Langmuir. Chaque système développé a démontré un potentiel contrôle du taux de relargage, ce qui en fait des candidats pour des investigations futures.

Study of medicinal and food plants as a source of biologically active compounds.

In the last few decades, scientific evidence has pointed out the health-beneficial effects of phenolic compounds in foods, including a decrease in risk of developing degenerative and chronic diseases, known to be caused by oxidative stress. In this frame can be inserted research carried out during my PhD thesis, which concerns the phytochemical investigation of phenolic composition in sweet cherries (Prunus avium L.), apple fruits (Malus domestica L.) and quinoa seeds (Chenopodium quinoa Willd.). The first project was focused on the investigation of phytochemical profile and nutraceutical value of fruits of new sweet cherry cultivars. Their phenolic profile and antioxidant activity were investigated and compared with those of commonly commercialized cultivars. Their nutraceutical value was evaluated in terms of antioxidant/neuroprotective capacity in neuron-like SH-SY5Y cells, in order to investigate their ability to counteract the oxidative stress and/or neurodegeneration process The second project was focused on phytochemical analysis of phenolic compounds in apples of ancient cultivars with the aim of selecting the most diverse cultivars, that will then be assayed for their anti-carcinogenic and anti-proliferative activities against the hepato-biliary and pancreatic tumours. The third project was focused on the analysis of polyphenolic pattern of seeds of two quinoa varieties grown at different latitudes. Analysis of phenolic profile and in vitro antioxidant activity of seed extracts both in their free and soluble-conjugated forms, showed that the accumulation of some classes of flavonoids is strictly regulated by environmental factors, even though the overall antioxidant capacity does not differ in quinoa Regalona grown in Chile and Italy. During the internship period carried out at the Department of Organic Chemistry at Universidad Autónoma de Madrid (UAM), it was achieved the isolation of two pentacyclic triterpenoids, from an endemic Peruvian plant, Jatropha macrantha Müll. Arg., with bio-guided fractionation technique.

Bio-preservation and bio-active compounds as sustainable strategies to improve quality and safety of fresh and fermented products

The development of new “green” and sustainable approaches to reduce food wastes, guaranteeing food quality, microbiological safety and the environmental sustainability, is of great interest for food industry. This PhD thesis, as part of the European project BioProMedFood (PRIMA–Section2 Programme), was focused on two strategies: the use of natural antimicrobials and the application of microbial strains isolated from spontaneously fermented products. The first part concerned the valorisation of microbial biodiversity of 15 Mediterranean spontaneously fermented sausages, through the isolation of autochthonous lactic acid bacteria (LAB) strains, mainly Latilactobacillus sakei, that were characterised regarding their safety and technological aspects. The most promising strains were tested as bio-protective cultures in fresh sausages, showing promising anti-listerial activity, or as starter cultures in fermented sausages. The second part of the research was focused on the use of natural compounds (phenolic extracts and essential oils from Juniperus oxycedrus needles and Rubus fruticosus leaves) with antimicrobial potential. They were tested in vitro against List. monocytogenes and Enterococcus faecium, showing differences in relation to species and type of extracts, but they hint at important possibilities for applications in specific foods. Concluding, this PhD thesis highlighted the great potential of traditional meat products as an isolation source of new strains with industrial importance. Moreover, the antimicrobial potential of compounds obtained from plant matrices opened promising perspectives to exploit them as “green” strategies to increase fresh food safety. The last topic of research, carry out in collaboration with Department of Nutrition and Food Sciences (University of Granada), investigated the effect of LAB fermentation on avocado leaves by-products, focusing on the bio-availability of phenolic compounds in the plant extracts, caused by microbial metabolism.

Biosurfactants produced by marine microorganisms with therapeutic applications

Marine microorganisms possess unique metabolic and physiological features and are an important source of new biomolecules, such as biosurfactants. Some of these surface-active compounds synthesized by marine microorganisms exhibit antimicrobial, anti-adhesive and anti-biofilm activity against a broad spectrum of human pathogens (including multi-drug resistant pathogens), and could be used instead of the existing drugs to treat infections caused by them. In other cases, these biosurfactants show anti-cancer activity, which could be envisaged as an alternative to conventional therapies. However, marine biosurfactants have not been widely explored, mainly due to the difficulties associated with the isolation and growth of their producing microorganisms. Culture-independent techniques (metagenomics) constitute a promising approach to study the genetic resources of otherwise inaccessible marine microorganisms without the requirement of culturing them, and can contribute to discover novel biosurfactants with significant biological activities. This paper reviews the most relevant biosurfactants produced by marine microorganisms with potential therapeutic applications and discusses future perspectives and opportunities to discover novel molecules from marine environments.

Effect of antifoams on oxygen mass transfer in pulp and paper mill effluents

Vaahdonestoaineiden haitallinen vaikutus hapen liukenemisnopeuteen biologisen puhdistamon jätevesissä on yleisesti tunnettua. Aineiden eri vaikutusmekanismien takia on silti vaikea etukäteen arvioida, miten ja kuinka paljon aineensiirto muuttuu. Työn tavoitteena oli saada tietoa vaahdonestoaineiden ja muiden pinta-aktiivisten aineiden vaikutuksesta kuplakokoon, kaasun tilavuusosuuteen ja kaasu-neste aineensiirtoon. Työn teoriaosassa on kuvailtu vaahdon muodostumiseen vaikuttavia tekijöitä sekä eri vaahdonestoaineiden vaikutusmekanismeja sellu- ja paperitehtaan jätevedessä. Edelleen on esitetty useita hapen siirtoa estäviä ja parantavia aineita. Työn kokeellisessa osassa tutkittiin kahdenkymmenenviiden eri pinta-aktiivisen aineen vaikutusta hapen liukenemisnopeuteen yksivaiheisessa kuplakolonnissa. Kokeet tehtiin kahdella pitoisuudella, kahdella eri kaasunjakolaitteella ja kolmella eri kaasun tyhjäputkinopeudella. Aineensiirtokokeiden rinnalla tutkittiin jätevesien laatu- ja fysikaalisiaominaisuuksia, niiden vaikutusta hapen liukenemisnopeuteen sekä testattavien koeaineiden vaikutusta fysikaalisin ominaisuuksiin. Kokeet osoittavat että pinta-aktiivisten aineiden vaikutus hapen aineensiirtoon vaihtelee riippuen kaasunjakolaitteesta ja aineen pitoisuuksista. Testatuista vaahdonestonaineista pienin negatiivinen vaikutus oli aineella AT 35 ja positiivinen vaikutustodettiin olevan vaahdonestoaineiden komponenteilla: P2, S1, F4 ja T9.

Biossurfactantes: propriedades e aplicações

Chemically synthesized surfactants are widely used for many purposes in almost every sector of modern industry. Surface-active compounds of biological origin (biosurfactants) have been gaining attention in recent years because of some advantages such as biodegradability, low toxicity, diversity of applications and functionality under extreme conditions. Microbial biosurfactants are useful in bioremediation of water and soil, enhanced oil recovery, and in many formulations of petrochemical, chemical, pharmaceutical, food, cosmetic and textile industries. The importance of biosurfactants, their characteristics and industrial applications are discussed.

Produção de biossurfactante por levedura

Biosurfactants are molecules extracellularly produced by bacteria, yeast and fungi that have significant interfacial activity properties. This review focuses on relevant parameters that influence biosurfactant production by yeasts. Many works have investigated the optimization of yeast biosurfactant production, mainly within the last decade, revealing that the potential of such microorganisms is not well explored in the industrial field. The main points to increase the process viability lays on the reduction of the production costs and enhancement of biosynthesis efficiency through optimization the culture conditions (carbon and nitrogen source, pH, aeration, speed agitation) and the selection of inexpensive medium components.