Acqua: solvente elettivo in Organocatalisi e Biocatalisi

Water is a safe, harmless, and environmentally benign solvent. From an eco-sustainable chemistry perspective, the use of water instead of organic solvent is preferred to decrease environmental contamination. Moreover, water has unique physical and chemical properties, such as high dielectric constant and high cohesive energy density compared to most organic solvents. The different interactions between water and substrates, make water an interesting candidate as a solvent or co-solvent from an industrial and laboratory perspective. In this regard, organic reactions in aqueous media are of current interest. In addition, from practical and synthetic standpoints, a great advantage of using water is immediately evident, since it does not require any preliminary drying process. This thesis was found on this aspect of chemical research, with particular attention to the mechanisms which control organo and bio-catalysis outcome. The first part of the study was focused on the aldol reaction. In particular, for the first time it has been analyzed for the first time the stereoselectivity of the condensation reaction between 3-pyridincarbaldehyde and the cyclohexanone, catalyzed by morpholine and 4-tertbutyldimethylsiloxyproline, using water as sole solvent. This interest has resulted in countless works appeared in the literature concerning the use of proline derivatives as effective catalysts in organic aqueous environment. These studies showed good enantio and diastereoselectivities but they did not present an in depth study of the reaction mechanism. The analysis of the products diastereomeric ratios through the Eyring equation allowed to compare the activation parameters (����H��� and ����S���) of the diastereomeric reaction paths, and to compare the different type of catalysis. While morpholine showed constant diasteromeric ratio at all temperatures, the O(TBS)-L-proline, showed a non-linear Eyring diagram, with two linear trends and the presence of an inversion temperature (Tinv) at 53 �� C, which denotes the presence of solvation effects by water. A pH-dependent study allowed to identify two different reaction mechanisms, and in the case of O(TBS)-L-proline, to ensure the formation of an enaminic species, as a keyelement in the stereoselective process. Moreover, it has been studied the possibility of using the 6- aminopenicillanic acid (6-APA) as amino acid-type catalyst for aldol condensation between cyclohexanone and aromatic aldehydes. A detailed analysis of the catalyst regarding its behavior in different organic solvents and pH, allowed to prove its potential as a candidate for green catalysis. Best results were obtained in neat conditions, where 6-APA proved to be an effective catalyst in terms of yields. The catalyst performance in terms of enantio- and diastereo-selectivity, was impaired by the competition between two different catalytic mechanisms: one via imine-enamine mechanism and one via a Bronsted-acid catalysis. The last part of the thesis was dedicated to the enzymatic catalysis, with particular attention to the use of an enzyme belonging to the class of alcohol dehydrogenase, the Horse Liver Alcohol Dehydrogenase (HLADH) which was selected and used in the enantioselective reduction of aldehydes to enantiopure arylpropylic alcohols. This enzyme has showed an excellent responsiveness to this type of aldehydes and a good tolerance toward organic solvents. Moreover, the fast keto-enolic equilibrium of this class of aldehydes that induce the stereocentre racemization, allows the dynamic-kinetic resolution (DKR) to give the enantiopure alcohol. By analyzing the different reaction parameters, especially the pH and the amount of enzyme, and adding a small percentage of organic solvent, it was possible to control all the parameters involved in the reaction. The excellent enatioselectivity of HLADH along with the DKR of arylpropionic aldehydes, allowed to obtain the corresponding alcohols in quantitative yields and with an optical purity ranging from 64% to >99%.

Enantioselective Reactions Promoted by Organocatalytic Species From The Natural Chiral Pool

During the course of my Ph.D. in the laboratories directed by Prof. Alfredo Ricci at the Department of Organic Chemistry ���A. Mangini��� of the University of Bologna, I was involved in the study and the application of a number of organocatalytic systems, all coming from the natural chiral pool. The first part of this thesis will be devoted to new homogeneous organocatalytic reactions promoted by Cinchona alkaloid-based organocatalysts. Quinine based catalysts were found to be a very effective catalyst for Diels-Alder reactions involving 3-vinylindoles. Excellent results in terms of yields and enantioselectivities were achieved, outlining also a remarkable organocatalytic operational mode mimicking enzymatic catalysis. The same reaction with 2-vinylindoles showed a completely different behaviour resulting in an unusual resolution-type process. The asymmetric formal [3+2] cycloaddition with in situ generated N-carbamoyl nitrones using Cinchona-derived quaternary ammonium salts as versatile catalysts under phase transfer conditions, outlines another application in organocatalysis of this class of alkaloids. During the seven months stage in the Prof. Helma Wennemers��� group at the Department of Chemistry of the University of Basel (Switzerland) I have been involved in organocatalysis promoted by oligopeptides. My contribution regarded the 1,4-addition reaction of aldehydes to nitroolefins. In the work performed at the Department of Organic Chemistry ���A. Mangini��� of the University of Bologna, in collaboration with the ���Institut Charles Gerhardt-Montpellier, of Montpellier (France) the possibility of performing for the first time heterogeneous organocatalysis by using a natural polysaccharide biopolymer as the source of chirality was disclosed. With chitosan, derived from deacetylation of chitin, a highly enantioselective heterogeneous organocatalytic aldol reaction could be performed. The use of an eco-friendly medium such as water, the recyclability of the catalytic specie and the renewable nature of the polysaccharide are assets of this new approach in organocatalysis and open interesting perspectives for the use of biopolymers.

Intrinsic uncoupling in the ATP synthase of Escherichia coli. Studies on WT and ����-truncated mutants

The H+/ATP ratio in the catalysis of ATP synthase has generally been considered a fixed parameter. However, Melandri and coworkers have recently shown that, in the ATP synthase of the photosynthetic bacterium Rb.capsulatus, this ratio can significantly decrease during ATP hydrolysis when the concentration of either ADP or Pi is maintained at a low level (Turina et al., 2004). The present work has dealt with the ATP synthase of E.coli, looking for evidence of this phenomenon of intrinsic uncoupling in this organism as well. First of all, we have shown that the DCCD-sensitive ATP hydrolysis activity of E.coli internal membranes was strongly inhibited by ADP and Pi, with a half-maximal effect in the submicromolar range for ADP and at 140 ����M for Pi. In contrast to this monotonic inhibition, however, the proton pumping activity of the enzyme, as estimated under the same conditions by the fluorescence quenching of the ����pH-sensitive probe ACMA, showed a clearly biphasic progression, both for Pi, increasing from 0 up to approximately 200 ����M, and for ADP, increasing from 0 up to a few ����M. We have interpreted these results as indicating that the occupancy of ADP and Pi binding sites shifts the enzyme from a partially uncoupled state to a fully coupled state, and we expect that the ADP- and Pi-modulated intrinsic uncoupling is likely to be a general feature of prokaryotic ATP synthases. Moreover, the biphasicity of the proton pumping data suggested that two Pi binding sites are involved. In order to verify whether the same behaviour could be observed in the isolated enzyme, we have purified the ATP synthase of E.coli and reconstituted it into liposomes. Similarly as observed in the internal membrane preparation, in the isolated and reconstituted enzyme it was possible to observe inhibition of the hydrolytic activity by ADP and Pi (with half-maximal effects at few ����M for ADP and at 400 ����M for Pi) with a concomitant stimulation of proton pumping. Both the inhibition of ATP hydrolysis and the stimulation of proton pumping as a function of Pi were lost upon ADP removal by an ADP trap. These data have made it possible to conclude that the results obtained in E.coli internal membranes are not due to the artefactual interference of enzymatic activities other than the ones of the ATP synthase. In addition, data obtained with liposomes have allowed a calibration of the ACMA signal by ����pH transitions of known extent, leading to a quantitative evaluation of the proton pumping data. Finally, we have focused our efforts on searching for a possible structural candidate involved in the phenomenon of intrinsic uncoupling. The ����-subunit of the ATP-synthase is known as an endogenous inhibitor of the hydrolysis activity of the complex and appears to undergo drastic conformational changes between a non-inhibitory form (down-state) and an inhibitory form (up-state)(Rodgers & Wilce, 2000; Gibbons et al., 2000). In addition, the results of Cipriano & Dunn (2006) indicated that the C-terminal domain of this subunit played an important role in the coupling mechanism of the pump, and those of Capaldi et al. (2001), Suzuki et al. (2003) were consistent with the down-state showing a higher hydrolysis-to-synthesis ratio than the up-state. Therefore, we decided to search for modulation of pumping efficiency in a C-terminally truncated ���� mutant. A low copy number expression vector has been built, carrying an extra copy of uncC, with the aim of generating an ����-overexpressing E.coli strain in which normal levels of assembly of the mutated ATP-synthase complex would be promoted. We have then compared the ATP hydrolysis and the proton pumping activity in membranes prepared from these ����-overexpressing E.coli strains, which carried either the WT ���� subunit or the ����88-stop truncated form. Both strains yielded well energized membranes. Noticeably, they showed a marked difference in the inhibition of hydrolysis by Pi, this effect being largely lost in the truncated mutant. However, pre-incubation of the mutated enzyme with ADP at low nanomolar concentrations (apparent Kd = 0.7nM) restored the hydrolysis inhibition, together with the modulation of intrinsic uncoupling by Pi, indicating that, contrary to wild-type, during membrane preparation the truncated mutant had lost the ADP bound at this high-affinity site, evidently due to a lower affinity (and/or higher release) for ADP of the mutant relative to wild type. Therefore, one of the effects of the C-terminal domain of ���� appears to be to modulate the affinity of at least one of the binding sites for ADP. The lack of this domain does not appear so much to influence the modulability of coupling efficiency, but instead the extent of this modulation. At higher preincubated ADP concentrations (apparent Kd = 117nM), the only observed effects were inhibition of both hydrolysis and synthesis, providing a direct proof that two ADP-binding sites on the enzyme are involved in the inhibition of hydrolysis, of which only the one at higher affinity also modulates the coupling efficiency.

Use of solvents and environmental friendly materials for applications in Green Chemistry

The present Thesis studies three alternative solvent groups as sustainable replacement of traditional organic solvents. Some aspects of fluorinated solvents, supercritical fluids and ionic liquids, have been analysed with a critical approach and their effective ���greenness��� has been evaluated from the points of view of the synthesis, the properties and the applications. In particular, the attention has been put on the environmental and human health issues, evaluating the eco-toxicity, the toxicity and the persistence, to underline that applicability and sustainability are subjects with equal importance. The ���green��� features of fluorous solvents and supercritical fluids are almost well-established; in particular supercritical carbon dioxide (scCO2) is probably the ���greenest��� solvent among the alternative solvent systems developed in the last years, enabling to combine numerous advantages both from the point of view of industrial/technological applications and eco-compatibility. In the Thesis the analysis of these two classes of alternative solvents has been mainly focused on their applicability, rather than the evaluation of their environmental impact. Specifically they have been evaluated as alternative media for non-aqueous biocatalysis. For this purpose, the hydrophobic ion pairing (HIP), which allows solubilising enzymes in apolar solvents by an ion pairing between the protein and a surfactant, has been investigated as effective enzymatic derivatisation technique to improve the catalytic activity under homogeneous conditions in non conventional media. The results showed that the complex enzyme-surfactant was much more active both in fluorous solvents and in supercritical carbon dioxide than the native form of the enzyme. Ionic liquids, especially imidazolium salts, have been proposed some years ago as ���fully green��� alternative solvents; however this epithet does not take into account several ���brown��� aspects such as their synthesis from petro-chemical starting materials, their considerable eco-toxicity, toxicity and resistance to biodegradation, and the difficulty of clearly outline applications in which ionic liquids are really more advantageous than traditional solvents. For all of these reasons in this Thesis a critical analysis of ionic liquids has been focused on three main topics: i) alternative synthesis by introducing structural moieties which could reduce the toxicity of the most known liquid salts, and by using starting materials from renewable resources; ii) on the evaluation of their environmental impact through eco-toxicological tests (Daphnia magna and Vibrio fischeri acute toxicity tests, and algal growth inhibition), toxicity tests (MTT test, AChE inhibition and LDH release tests) and fate and rate of aerobic biodegradation in soil and water; iii) and on the demonstration of their effectiveness as reaction media in organo-catalysis and as extractive solvents in the recovery of vegetable oil from terrestrial and aquatic biomass. The results about eco-toxicity tests with Daphnia magna, Vibrio fischeri and algae, and toxicity assay using cultured cell lines, clearly indicate that the difference in toxicity between alkyl and oxygenated cations relies in differences of polarity, according to the general trend of decreasing toxicity by decreasing the lipophilicity. Independently by the biological approach in fact, all the results are in agreement, showing a lower toxicity for compounds with oxygenated lateral chains than for those having purely alkyl lateral chains. These findings indicate that an appropriate choice of cation and anion structures is important not only to design the IL with improved and suitable chemico-physical properties but also to obtain safer and eco-friendly ILs. Moreover there is a clear indication that the composition of the abiotic environment has to be taken into account when the toxicity of ILs in various biological test systems is analysed, because, for example, the data reported in the Thesis indicate a significant influence of salinity variations on algal toxicity. Aerobic biodegradation of four imidazolium ionic liquids, two alkylated and two oxygenated, in soil was evaluated for the first time. Alkyl ionic liquids were shown to be biodegradable over the 6 months test period, and in contrast no significant mineralisation was observed with oxygenated derivatives. A different result was observed in the aerobic biodegradation of alkylated and oxygenated pyridinium ionic liquids in water because all the ionic liquids were almost completely degraded after 10 days, independently by the number of oxygen in the lateral chain of the cation. The synthesis of new ionic liquids by using renewable feedstock as starting materials, has been developed through the synthesis of furan-based ion pairs from furfural. The new ammonium salts were synthesised in very good yields, good purity of the products and wide versatility, combining low melting points with high decomposition temperatures and reduced viscosities. Regarding the possible applications as surfactants and biocides, furan-based salts could be a valuable alternative to benzyltributylammonium salts and benzalkonium chloride that are produced from non-renewable resources. A new procedure for the allylation of ketones and aldehydes with tetraallyltin in ionic liquids was developed. The reaction afforded high yields both in sulfonate-containing ILs and in ILs without sulfonate upon addition of a small amount of sulfonic acid. The checked reaction resulted in peculiar chemoselectivity favouring aliphatic substrates towards aromatic ketones and good stereoselectivity in the allylation of levoglucosenone. Finally ILs-based systems could be easily and successfully recycled, making the described procedure environmentally benign. The potential role of switchable polarity solvents as a green technology for the extraction of vegetable oil from terrestrial and aquatic biomass has been investigated. The extraction efficiency of terrestrial biomass rich in triacylglycerols, as soy bean flakes and sunflower seeds, was comparable to those of traditional organic solvents, being the yield of vegetable oils recovery very similar. Switchable polarity solvents as been also exploited for the first time in the extraction of hydrocarbons from the microalga Botryococcus braunii, demonstrating the efficiency of the process for the extraction of both dried microalgal biomass and directly of the aqueous growth medium. The switchable polarity solvents exhibited better extraction efficiency than conventional solvents, both with dried and liquid samples. This is an important issue considering that the harvest and the dewatering of algal biomass have a large impact on overall costs and energy balance.

N-Heterocyclic carbene complexes of rhodium: structures, dynamics and catalysis

A series of imidazolium salts of the type [BocNHCH2CH2ImR]X (Boc = t-Bu carbamates; Im = imidazole) (R = Me, X = I, 1a; R = Bn, X = Br, 1b; R = Trityl, X = Cl, 1c) and [BnImR���]X (R��� = Me, X = Br, 1d; R��� = Bn, X = Br, 1e; R��� = Trityl, X = Cl, 1g; R��� = tBu, X = Br, 1h) bearing increasingly bulky substituents were synthetized and characterized. Subsequently, these precursors were employed in the synthesis of silver(I)-N-heterocyclic (NHC) complexes as transmetallating reagents for the preparation of rhodium(I) complexes [RhX(NBD)(NHC)] (NHC = 1-(2-NHBoc-ethyl)-3-R-imidazolin-2-ylidene; X = Cl; R = Me, 4a; R = Bn, 4b; R = Trityl, 4c; X = I, R = Me, 5a; NHC = 1-Bn-3-R���-imidazolin-2-ylidene; X = Cl; R��� = Me, 4d, R��� = Bn, 4e, R��� = Trityl, 4g; R��� = tBu, 4h). VT NMR studies of these complexes revealed a restricted rotation barriers about the metal-carbene bond. While the rotation barriers calculated for the complexes in which R = Me, Bn (4a,b,d,e and 5a) matched the experimental values, this was not true for the complexes 4c,g, bearing a trityl group for which the values are much smaller than the calculated ones. Energy barriers for 4c,g, derived from a line shape simulation, showed a strong dependence on the temperature while for 4h the rotational energy barrier is stopped at room temperature. The catalytic activity of the new rhodium compounds was investigated in the hydrosilylation of terminal alkynes and in the addition of phenylboronic acid to benzaldehyde. The imidazolium salts 1d,e were also employed in the synthesis of new iron(II)-NHC complexes. Finally, during a six-months stay at the University of York a new ligand derived from Norharman was prepared and employed in palladium-mediated cross-coupling.