Processi di biorefining per l'estrazione di secondary chemical building blocks da sottoprotti dell'agro-industria

Phenol and cresols represent a good example of primary chemical building blocks of which 2.8 million tons are currently produced in Europe each year. Currently, these primary phenolic building blocks are produced by refining processes from fossil hydrocarbons: 5% of the world-wide production comes from coal (which contains 0.2% of phenols) through the distillation of the tar residue after the production of coke, while 95% of current world production of phenol is produced by the distillation and cracking of crude oil. In nature phenolic compounds are present in terrestrial higher plants and ferns in several different chemical structures while they are essentially absent in lower organisms and in animals. Biomass (which contain 3-8% of phenols) represents a substantial source of secondary chemical building blocks presently underexploited. These phenolic derivatives are currently used in tens thousand of tons to produce high cost products such as food additives and flavours (i.e. vanillin), fine chemicals (i.e. non-steroidal anti-inflammatory drugs such as ibuprofen or flurbiprofen) and polymers (i.e. poly p-vinylphenol, a photosensitive polymer for electronic and optoelectronic applications). European agrifood waste represents a low cost abundant raw material (250 millions tons per year) which does not subtract land use and processing resources from necessary sustainable food production. The class of phenolic compounds is essentially constituted by simple phenols, phenolic acids, hydroxycinnamic acid derivatives, flavonoids and lignans. As in the case of coke production, the removal of the phenolic contents from biomass upgrades also the residual biomass. Focusing on the phenolic component of agrifood wastes, huge processing and marketing opportunities open since phenols are used as chemical intermediates for a large number of applications, ranging from pharmaceuticals, agricultural chemicals, food ingredients etc. Following this approach we developed a biorefining process to recover the phenolic fraction of wheat bran based on enzymatic commercial biocatalysts in completely water based process, and polymeric resins with the aim of substituting secondary chemical building blocks with the same compounds naturally present in biomass. We characterized several industrial enzymatic product for their ability to hydrolize the different molecular features that are present in wheat bran cell walls structures, focusing on the hydrolysis of polysaccharidic chains and phenolics cross links. This industrial biocatalysts were tested on wheat bran and the optimized process allowed to liquefy up to the 60 % of the treated matter. The enzymatic treatment was also able to solubilise up to the 30 % of the alkali extractable ferulic acid. An extraction process of the phenolic fraction of the hydrolyzed wheat bran based on an adsorbtion/desorption process on styrene-polyvinyl benzene weak cation-exchange resin Amberlite IRA 95 was developed. The efficiency of the resin was tested on different model system containing ferulic acid and the adsorption and desorption working parameters optimized for the crude enzymatic hydrolyzed wheat bran. The extraction process developed had an overall yield of the 82% and allowed to obtain concentrated extracts containing up to 3000 ppm of ferulic acid. The crude enzymatic hydrolyzed wheat bran and the concentrated extract were finally used as substrate in a bioconversion process of ferulic acid into vanillin through resting cells fermentation. The bioconversion process had a yields in vanillin of 60-70% within 5-6 hours of fermentation. Our findings are the first step on the way to demonstrating the economical feasibility for the recovery of biophenols from agrifood wastes through a whole crop approach in a sustainable biorefining process.

Organocatalytic asymmetric mannich-type reactions: an easy approach to optically active amine derivatives

The topics I came across during the period I spent as a Ph.D. student are mainly two. The first concerns new organocatalytic protocols for Mannich-type reactions mediated by Cinchona alkaloids derivatives (Scheme I, left); the second topic, instead, regards the study of a new approach towards the enantioselective total synthesis of Aspirochlorine, a potent gliotoxin that recent studies indicate as a highly selective and active agent against fungi (Scheme I, right). At the beginning of 2005 I had the chance to join the group of Prof. Alfredo Ricci at the Department of Organic Chemistry of the University of Bologna, starting my PhD studies. During the first period I started to study a new homogeneous organocatalytic aza-Henry reaction by means of Cinchona alkaloid derivatives as chiral base catalysts with good results. Soon after we introduced a new protocol which allowed the in situ synthesis of N-carbamoyl imines, scarcely stable, moisture sensitive compounds. For this purpose we used α-amido sulfones, bench stable white crystalline solids, as imine precursors (Scheme II). In particular we were able to obtain the aza-Henry adducts, by using chiral phase transfer catalysis, with a broad range of substituents as R-group and excellent results, unprecedented for Mannich-type transformations (Scheme II). With the optimised protocol in hand we have extended the methodology to the other Mannich-type reactions. We applied the new method to the Mannich, Strecker and Pudovik (hydrophosphonylation of imines) reactions with very good results in terms of enantioselections and yields, broadening the usefulness of this novel protocol. The Mannich reaction was certainly the most extensively studied work in this thesis (Scheme III). Initially we developed the reaction with α-amido sulfones as imine precursors and non-commercially available malonates with excellent results in terms of yields and enantioselections.3 In this particular case we recorded 1 mol% of catalyst loading, very low for organocatalytic processes. Then we thought to develop a new Mannich reaction by using simpler malonates, such as dimethyl malonate.4 With new optimised condition the reaction provided slightly lower enantioselections than the previous protocol, but the Mannich adducts were very versatile for the obtainment of β3-amino acids. Furthermore we performed the first addition of cyclic β-ketoester to α-amido sulfones obtaining the corresponding products in good yield with high level of diastereomeric and enantiomeric excess (Scheme III). Further studies were done about the Strecker reaction mediated by Cinchona alkaloid phase-transfer quaternary ammonium salt derivatives, using acetone cyanohydrin, a relatively harmless cyanide source (Scheme IV). The reaction proceeded very well providing the corresponding α-amino nitriles in good yields and enantiomeric excesses. Finally, we developed two new complementary methodologies for the hydrophosphonylation of imines (Scheme V). As a result of the low stability of the products derived from aromatic imines, we performed the reactions in mild homogeneous basic condition by using quinine as a chiral base catalyst giving the α-aryl-α-amido phosphonic acid esters as products (Scheme V, top).6 On the other hand, we performed the addition of dialkyl phosphite to aliphatic imines by using chiral Cinchona alkaloid phase transfer quaternary ammonium salt derivatives using our methodology based on α-amido sulfones (Scheme V, bottom). The results were good for both procedures covering a broad range of α-amino phosphonic acid ester. During the second year Ph.D. studies, I spent six months in the group of Prof. Steven V. Ley, at the Department of Chemistry of the University of Cambridge, in United Kingdom. During this fruitful period I have been involved in a project concerning the enantioselective synthesis of Aspirochlorine. We provided a new route for the synthesis of a key intermediate, reducing the number of steps and increasing the overall yield. Then we introduced a new enantioselective spirocyclisation for the synthesis of a chiral building block for the completion of the synthesis (Scheme VI).

Determination of bioactive and potentially toxic compounds in food: raw material analysis and shelf life evaluation

"Bioactive compounds" are extranutritional constituents that typically occur in small quantities in food. They are being intensively studied to evaluate their effects on health. Bioactive compounds include both water soluble compounds, such as phenolics, and lipidic substances such as n-3 fatty acids, tocopherols and sterols. Phenolic compounds, tocopherols and sterols are present in all plants and have been studied extensively in cereals, nuts and oil. n-3 fatty acids are present in fish and all around the vegetable kingdom. The aim of the present work was the determination of bioactive and potentially toxic compounds in cereal based foods and nuts. The first section of this study was focused on the determination of bioactive compounds in cereals. Because of that the different forms of phytosterols were investigated in hexaploid and tetraploid wheats. Hexaploid cultivars were the best source of esterified sterols (40.7% and 37.3% of total sterols for Triticum aestivum and Triticum spelta, respectively). Significant amounts of free sterols (65.5% and 60.7% of total sterols for Triticum durum and Triticum dicoccon, respectively) were found in the tetraploid cultivars. Then, free and bound phenolic compounds were identified in barley flours. HPLCESI/ MSD analysis in negative and positive ion mode established that barley free flavan-3- ols and proanthocyanidins were four dimers and four trimers having (epi)catechin and/or (epi)gallocatechin (C and/or GC) subunits. Hydroxycinnamic acids and their derivatives were the main bound phenols in barley flours. The results obtained demonstrated that barley flours were rich in phenolic compounds that showed high antioxidant activity. The study also examined the relationships between phenolic compounds and lipid oxidation of bakery. To this purpose, the investigated barley flours were used in the bakery production. The formulated oven products presented an interesting content of phenolic compounds, but they were not able to contain the lipid oxidation. Furthermore, the influence of conventional packaging on lipid oxidation of pasta was evaluated in n-3 enriched spaghetti and egg spaghetti. The results proved that conventional packaging was not appropriated to preserve pasta from lipid oxidation; in fact, pasta that was exposed to light showed a high content of potentially toxic compounds derived from lipid oxidation (such as peroxide, oxidized fatty acids and COPs). In the second section, the content of sterols, phenolic compounds, n-3 fatty acids and tocopherols in walnuts were reported. Rapid analytical techniques were used to analyze the lipid fraction and to characterize phenolic compounds in walnuts. Total lipid chromatogram was used for the simultaneous determination of the profile of sterols and tocopherols. Linoleic and linolenic acids were the most representative n-6 and n-3 essential dietary fatty acids present in these nuts. Walnuts contained substantial amounts of γ- and δ-tocopherol, which explained their antioxidant properties. Sitosterol, Δ5-avenasterol and campesterol were the major free sterols found. Capillary electrophoresis coupled to DAD and microTOF was utilized to determine phenolic content of walnut. A new compound in walnut ((2E,4E)- 8-hydroxy-2,7-dimethyl-2,4-decadiene-1,10-dioic acid 6-O-β-D-glucopiranosyl ester, [M−H]− 403.161m/z) with a structure similar to glansreginins was also identified. Phenolic compounds corresponded to 14–28% of total polar compounds quantified. Aglycone and glycosylated ellagic acid represented the principal components and account for 64–75% of total phenols in walnuts. However, the sum of glansreginins A, B and ((2E,4E)-8-hydroxy- 2,7-dimethyl-2,4-decadiene-1,10-dioic acid 6-O-β-D-glucopiranosyl ester was in the range of 72–86% of total quantified compounds.

Reactivity of activated electrophiles and nucleophiles: labile intermediates and properties of the reaction products

The main topic of my Ph.D. thesis is the study of nucleophilic and electrophilic aromatic substitution reaction, in particular from a mechanistic point of view. The research was mainly focused on the reactivity of superactivated aromatic systems. In spite of their high reactivity (hence the high reaction’s rate), we were able to identify and in some case to isolate -complexes until now only hypothesized. For example, interesting results comes from the study of the protonation of the supernucleophiles tris(dialkylamino)benzenes. However, the best result obtained in this field was the isolation and structural characterization of the first stables zwitterionic Wheland-Meisenheimer complexes by using 2,4-dipyrrolidine-1,3-thiazole as supernucleophile and 4,6-dinitrobenzofuroxan or 4,6-dinitrotetrazolepyridine as superelectrophile. These reactions were also studied by means of computational chemistry, which allowed us to better investigate on the energetic and properties of the reactions and reactants studied. We also discovered, in some case fortuitously, some relevant properties and application of the compounds we synthesized, such as fluorescence in solid state and nanoparticles, or textile dyeing. We decided to investigate all these findings also by collaborating with other research groups. During a period in the “Laboratoire de Structure et Réactivité des Systèmes Moléculaires Complexes-SRSMC, Université de Lorraine et CNRS, France, I carried out computational studies on new iron complexes for the use as dyes in Dye Sensitized Solar Cells (DSSC). Furthermore, thanks to this new expertise, I was involved in a collaboration for the study of the ligands’ interaction in biological systems. A collaboration with University of Urbino allowed us to investigate on the reactivity of 1,2-diaza-1,3-dienes toward nucleophiles such as amino and phosphine derivatives, which led to the synthesis of new products some of which are 6 or 7 member heterocycles containing both phosphorus and nitrogen atoms.