Polysulfurated aromatic compounds: preparation and photophysical properties

The aim of this thesis was the synthesis and photophysical characterization of some new polysulfurated aromatic compounds: this class of molecules can offer intriguing properties, potentially useful for the construction of new materials for optoelectronic devices. Two main families of compounds have been synthesized: the first is represented by a series of small molecular asterisks, with peripheral aromatic units, showing luminescence in solid phase or in highly rigid conditions. All compounds with peripheral substituents display an AIE behavior (Aggregation Induced Emission) with radiative deactivation of the triplet states. Taking inspiration from these smaller asterisks, a larger molecule with the same geometry has been designed, decorated with terpyridyl moieties as the outermost units: this compound shows great affinity for the coordination of several transition metal ions, changing luminescence properties after the interaction with zinc ions. With the same intentions, a tetrasulfurated pyrene-core molecule with terpyridyl external units has been synthesized and isolated: this ligand exhibits good coordination capabilities towards transition metal ions, giving rise to luminescent nanoaggregates upon addition of zinc(II), characterized by DLS and AFM microscopy. In addition a NIR emission is recorded after coordination of neodymium(III), showing evidence of an intramolecular energy transfer process.

Geological and hydrogeological features affecting migration, multi-phase partitioning and degradation of chlorinated hydrocarbons through unconsolidated porous media.

Chlorinated solvents are the most ubiquitous organic contaminants found in groundwater since the last five decades. They generally reach groundwater as Dense Non-Aqueous Phase Liquid (DNAPL). This phase can migrate through aquifers, and also through aquitards, in ways that aqueous contaminants cannot. The complex phase partitioning to which chlorinated solvent DNAPLs can undergo (i.e. to the dissolved, vapor or sorbed phase), as well as their transformations (e.g. degradation), depend on the physico-chemical properties of the contaminants themselves and on features of the hydrogeological system. The main goal of the thesis is to provide new knowledge for the future investigations of sites contaminated by DNAPLs in alluvial settings, proposing innovative investigative approaches and emphasizing some of the key issues and main criticalities of this kind of contaminants in such a setting. To achieve this goal, the hydrogeologic setting below the city of Ferrara (Po plain, northern Italy), which is affected by scattered contamination by chlorinated solvents, has been investigated at different scales (regional and site specific), both from an intrinsic (i.e. groundwater flow systems) and specific (i.e. chlorinated solvent DNAPL behavior) point of view. Detailed investigations were carried out in particular in one selected test-site, known as “Caretti site”, where high-resolution vertical profiling of different kind of data were collected by means of multilevel monitoring systems and other innovative sampling and analytical techniques. This allowed to achieve a deep geological and hydrogeological knowledge of the system and to reconstruct in detail the architecture of contaminants in relationship to the features of the hosting porous medium. The results achieved in this thesis are useful not only at local scale, e.g. employable to interpret the origin of contamination in other sites of the Ferrara area, but also at global scale, in order to address future remediation and protection actions of similar hydrogeologic settings.

Microbial ecology of biotechnological processes

The investigation of phylogenetic diversity and functionality of complex microbial communities in relation to changes in the environmental conditions represents a major challenge of microbial ecology research. Nowadays, particular attention is paid to microbial communities occurring at environmental sites contaminated by recalcitrant and toxic organic compounds. Extended research has evidenced that such communities evolve some metabolic abilities leading to the partial degradation or complete mineralization of the contaminants. Determination of such biodegradation potential can be the starting point for the development of cost effective biotechnological processes for the bioremediation of contaminated matrices. This work showed how metagenomics-based microbial ecology investigations supported the choice or the development of three different bioremediation strategies. First, PCR-DGGE and PCR-cloning approaches served the molecular characterization of microbial communities enriched through sequential development stages of an aerobic cometabolic process for the treatment of groundwater contaminated by chlorinated aliphatic hydrocarbons inside an immobilized-biomass packed bed bioreactor (PBR). In this case the analyses revealed homogeneous growth and structure of immobilized communities throughout the PBR and the occurrence of dominant microbial phylotypes of the genera Rhodococcus, Comamonas and Acidovorax, which probably drive the biodegradation process. The same molecular approaches were employed to characterize sludge microbial communities selected and enriched during the treatment of municipal wastewater coupled with the production of polyhydroxyalkanoates (PHA). Known PHA-accumulating microorganisms identified were affiliated with the genera Zooglea, Acidovorax and Hydrogenophaga. Finally, the molecular investigation concerned communities of polycyclic aromatic hydrocarbon (PAH) contaminated soil subjected to rhizoremediation with willow roots or fertilization-based treatments. The metabolic ability to biodegrade naphthalene, as a representative model for PAH, was assessed by means of stable isotope probing in combination with high-throughput sequencing analysis. The phylogenetic diversity of microbial populations able to derive carbon from naphthalene was evaluated as a function of the type of treatment.

The quest for planarizing distortions in hydrocarbons: two stereoisomeric 4.5.5.5 fenestranes

According to semiempirical calculations the planarizing distortions in the central C(C)4 substructure of fenestranes, represented as 1, can be enhanced by a variety of structural modifications. Based on these results we selected the 7-hydroxy-c,c,c,c- and c,t,c,c[4.5.5.5]fenestranones 13 and 16 as precursors for the introduction of a bridgehead double bond. The efficient synthesis of these precursors and their chemical transformations are reported. Attempts to activate the hydroxyl group in 16 for introduction of a bridgehead double bond led to the rearrangement of the [4.5.5.5]fenestrane to a triquinacane skeleton. (C) 2011 Elsevier Ltd. All rights reserved.

Aspiration toxicology of hydrocarbons and lamp oils studied by in vitro technology

Medical literature regularly reports on accidental poisoning in children after aspiration of combustibles such as lamp oils which usually contain hydrocarbons or rape methyl esters (RMEs). We aimed to analyze the toxic potential of alkanes and different combustible classes in vitro with regard to biologic responses and mechanisms mediating toxicity. Two different in vitro models were used, i.e. (i) a captive bubble surfactometer (CBS) to assess direct influence of combustibles on biophysical properties of surfactant film and (ii) cell cultures (BEAS-2B and R3/1 cells, primary macrophages, re-differentiated epithelia) closely mimicking the inner lung surface. Biological endpoints included cell viability, cytotoxicity and inflammatory mediator release. CBS measurements demonstrate that combustibles affect film dynamics, i.e. the surface tension/area characteristics during compression and expansion, in a dose and molecular chain length dependent manner. Cell culture results confirm the dose dependent toxicity. Generally, cytotoxicity and cytokine release are higher in short-chained alkanes and hydrocarbon-based combustibles than in long-chained substances, e.g. highest inducible cytotoxicity in BEAS-2B was for hexane 84.6%, decane 74% and hexadecane 30.8%. Effects of RME-based combustibles differed between the cell models. Our results confirm data from animal experiments and give new insights into the mechanisms underlying the adverse health effects observed.

Comparative aromatic hydroxylation and N-demethylation of MPTP neurotoxin and its analogs, N-methylated beta-carboline and isoquinoline alkaloids, by human cytochrome P450 2D6

1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) neurotoxin is a chemical inducer of Parkinson's disease (PD) whereas N-methylated beta-carbolines and isoquinolines are naturally occurring analogues of MPTP involved in PD. This research has studied the oxidation of MPTP by human CYP2D6 (CYP2D6*1 and CYP2D6*10 allelic variants) as well as by a mixture of cytochrome P450s-resembling HLM, and the products generated compared with those afforded by human monoamine oxidase (MAO-B). MPTP was efficiently oxidized by CYP2D6 to two main products: MPTP-OH (p-hydroxylation) and PTP (N-demethylation), with turnover numbers of 10.09 min-1 and Km of 79.36+/-3 microM (formation of MPTP-OH) and 18.95 min-1 and Km 69.6+/-2.2 microM (PTP). Small amounts of dehydrogenated toxins MPDP+ and MPP+ were also detected. CYP2D6 competed with MAO-B for the oxidation of MPTP. MPTP oxidation by MAO-B to MPDP+ and MPP+ toxins (bioactivation) was up to 3-fold higher than CYP2D6 detoxification to PTP and MPTP-OH. Several N-methylated beta-carbolines and isoquinolines were screened for N-demethylation (detoxification) that was not significantly catalyzed by CYP2D6 or the P450s mixture. In contrast, various beta-carbolines were efficiently hydroxylated to hydroxy-beta-carbolines by CYP2D6. Thus, N(2)-methyl-1,2,3,4-tetrahydro-beta-carboline (a close MPTP analog) was highly hydroxylated to 6-hydroxy-N(2)-methyl-1,2,3,4-tetrahydro-beta-carboline and a corresponding 7-hydroxy-derivative. Thus, CYP2D6 could participate in the bioactivation and/or detoxification of these neuroactive compounds by an active hydroxylation pathway. The CYP2D6*1 enzymatic variant exhibited much higher metabolism of both MPTP and N(2)-methyl-1,2,3,4-tetrahydro-beta-carboline than the CYP2D6*10 variant, highlighting the importance of CYP2D6 polymorphism in the oxidation of these toxins. Altogether, these results suggest that CYP2D6 can play an important role in the metabolic outcome of both MPTP and beta-carbolines.

Molecular characterization of aromatic peroxygenase from Agrocybe aegerita

Recently, a novel group of fungal peroxidases, known as the aromatic peroxygenases (APO), has been discovered. Members of these extracellular biocatalysts produced by agaric basidiomycetes such as Agrocybe aegerita or Coprinellus radians catalyze reactions--for example, the peroxygenation of naphthalene, toluene, dibenzothiophene, or pyridine--which are actually attributed to cytochrome P450 monooxygenases. Here, for the first time, genetic information is presented on this new group of peroxide-consuming enzymes. The gene of A. aegerita peroxygenase (apo1) was identified on the level of messenger RNA and genomic DNA. The gene sequence was affirmed by peptide sequences obtained through an Edman degradation and de novo peptide sequencing of the purified enzyme. Quantitative real-time reverse transcriptase polymerase chain reaction demonstrated that the course of enzyme activity correlated well with that of mRNA signals for apo1 in A. aegerita. The full-length sequences of A. aegerita peroxygenase as well as a partial sequence of C. radians peroxygenase confirmed the enzymes' affiliation to the heme-thiolate proteins. The sequences revealed no homology to classic peroxidases, cytochrome P450 enzymes, and only little homology (<30%) to fungal chloroperoxidase produced by the ascomycete Caldariomyces fumago (and this only in the N-terminal part of the protein comprising the heme-binding region and part of the distal heme pocket). This fact reinforces the novelty of APO proteins. On the other hand, homology retrievals in genetic databases resulted in the identification of various APO homologous genes and transcripts, particularly among the agaric fungi, indicating APO's widespread occurrence in the fungal kingdom.

Hydroxylation of naphthalene by aromatic peroxygenase from Agrocybe aegerita proceeds via oxygen transfer from H2O2 and intermediary epoxidation

Agrocybe aegerita peroxidase/peroxygenase (AaP) is an extracellular fungal biocatalyst that selectively hydroxylates the aromatic ring of naphthalene. Under alkaline conditions, the reaction proceeds via the formation of an intermediary product with a molecular mass of 144 and a characteristic UV absorption spectrum (A(max) 210, 267, and 303 nm). The compound was semistable at pH 9 but spontaneously hydrolyzed under acidic conditions (pH<7) into 1-naphthol as major product and traces of 2-naphthol. Based on these findings and literature data, we propose naphthalene 1,2-oxide as the primary product of AaP-catalyzed oxygenation of naphthalene. Using (18)O-labeled hydrogen peroxide, the origin of the oxygen atom transferred to naphthalene was proved to be the peroxide that acts both as oxidant (primary electron acceptor) and oxygen source.

A Simple Algorithm for Unique Representation of Chemical Structures - Cyclic/ Acyclic Functionalized Achiral Hydrocarbons

An algorithm, based on ‘vertex priority values’ has been proposed to uniquely sequence and represent connectivity matrix of chemical structures of cyclic/ acyclic functionalized achiral hydrocarbons and their derivatives. In this method ‘vertex priority values’ have been assigned in terms of atomic weights, subgraph lengths, loops, and heteroatom contents. Subsequently the terminal vertices have been considered upon completing the sequencing of the core vertices. This approach provides a multilayered connectivity graph, which can be put to use in comparing two or more structures or parts thereof for any given purpose. Furthermore the basic vertex connection tables generated here are useful in the computation of characteristic matrices/ topological indices, automorphism groups, and in storing, sorting and retrieving of chemical structures from databases.