Preparation of new materials by heterogeneous modification of cellulose

O presente trabalho teve como principal objectivo estudar a modificação química heterogénea controlada de fibras de celulose com diferentes reagentes de modo a alterar as suas propriedades de superfície, em especial em termos da criação de um carácter hidrofóbico e lipofóbico, preservando, sempre que possível, as suas propriedades mecânicas e, consequentemente, abrindo novas perspectivas de aplicação. O desenvolvimento do trabalho envolveu três abordagens principais, envolvendo, em cada caso, o estudo de diferentes condições reaccionais. Na primeira abordagem foram utilizados como reagentes de modificação compostos perfluorados, nomeadamente o anidrido trifluoroacético (TFAA), o cloreto de 2,3,4,5,6-pentafluorobenzoílo (PFBz) e o cloreto de 3,3,3- trifluoropropanoílo (TFP), para promover a acilação heterogénea da superfície das fibras. A segunda estratégia usada consistiu na preparação de híbridos de celulose do tipo orgânico-inorgânico classe-II, através da modificação das fibras de celulose com o (3-isocianatopropil)trietoxissilano (ICPTEOS), um reagente organossilano bifuncional. A ligação às fibras de celulose foi efectuada através das funções isocianato e, posteriormente, os grupos etoxissilano foram sujeitos a tratamentos de hidrólise ácida, como tal ou na presença de outros siloxanos, nomeadamente o tetraetoxissilano (TEOS) e o 1H,1H,2H,2Hperfluorodeciltrietoxissilano (PFDTEOS). Finalmente, a última abordagem foi baseada na modificação das fibras com triclorometilssilano (TCMS), através de uma reacção gás-sólido, que dispensou assim o uso de solventes orgânicos. A ocorrência de modificação química foi em cada caso confirmada por Espectroscopia de Infravermelho com Transformada de Fourier e Reflectância Total Atenuada (FTIR-ATR), Análise Elementar (EA) e determinação de ângulos de contacto. Adicionalmente, e dependendo de cada caso específico, diversas outras técnicas foram empregues na caracterização aprofundada dos materiais preparados, nomeadamente Ressonância Magnética Nuclear CPMAS no Estado Sólido (RMN), Espectroscopia de Difracção de Raios-X (XRD), Análise Termogravimétrica (TGA), Espectrometria de Massa de Iões Secundários com Análise de Tempo de Vôo (ToF-SIMS), Espectroscopia Fotoelectrónica de Raios-X (XPS) e Microscopia Electrónica de Varrimento (SEM). Relativamente à acilação das fibras de celulose com reagentes perfluorados, o sucesso da reacção foi comprovado por FTIR-ATR, EA, XPS e ToF-SIMS. Neste contexto, obtiveram-se fibras modificadas possuindo graus de substituição (DS) compreendidos entre 0.006 e 0.39. Verificou-se por XRD que, em geral, mesmo para os valores de DS mais elevados, a cristalinidade das fibras não foi afectada, indicando que a modificação foi limitada às camadas mais superficiais das mesmas ou a regiões amorfas das suas camadas mais internas. Adicionalmente, observou-se por ToF-SIMS que a distribuição dos grupos perfluorados à superfície das fibras foi, de facto, bastante heterogénea. Todos os derivados de celulose perfluorados apresentaram elevada hidrofobicidade e lipofobicidade, tendo-se atingido ângulos de contacto com água e diiodometano de 126º e 104º, respectivamente. Um aspecto interessante relativo a estes materiais é que a elevada omnifobicidade foi observada mesmo para valores de DS muito reduzidos, não se mostrando significativamente afectada pelo aumento dos mesmos. Em consonância, verificou-se por XPS que a cobertura da superfície das fibras de celulose com grupos perfluorados aumentou apenas ligeiramente com o aumento do DS, apontando para a esterificação de camadas mais internas das fibras, associada, neste caso, predominantemente aos seus domínios amorfos. No que diz respeito à estabilidade hidrolítica destes derivados, obtiveram-se dois tipos distintos de comportamento. Por um lado, as fibras de celulose trifluoroacetiladas são facilmente hidrolisáveis em meio neutro, e, por outro, as fibras pentafluorobenzoiladas e trifluoropropanoiladas mostram-se bastante resistentes face a condições de hidrólise em meio neutro e ácido (pH 4), podendo, contudo, ser facilmente hidrolisadas em meio alcalino (pH 9 e 12, para derivados do PFBz e do TFP, respectivamente). Na segunda abordagem verificou-se a ocorrência de reacção por FTIR-ATR e EA. Em geral, a modificação química com ICPTEOS ocorre predominantemente nas zonas mais superficiais das fibras de celulose ou em regiões amorfas. Contudo, em condições reaccionais mais severas (maior quantidade de reagente e tempo de reacção), esta atingiu também regiões cristalinas, afectando, consequentemente, a estrutura cristalina das fibras, como verificado por XRD. Por RMN de 29Si observou-se que após reacção com o ICPTEOS já existiam indícios de alguma hidrólise dos grupos etoxissilano, e que a sua subsequente condensação parcial tinha levado à formação de uma película inorgânica em redor das fibras (verificado por SEM), constituída maioritariamente por estruturas lineares, com uma contribuição mais modesta de estruturas “diméricas” e outras mais ramificadas. Consequentemente, este revestimento inorgânico transformou as fibras de celulose em materiais híbridos com elevada hidrofobicidade (ângulos de contacto com água entre 103-129º). A hidrólise ácida dos restantes grupos etoxissilano, como tal ou na presença de TEOS, originou híbridos de celulose com elevada hidrofilicidade, sendo impossível medirem-se os ângulos de contacto com água dos produtos finais, devido à presença maioritária de grupos silanol (Si-OH) e ligações Si-O-Si à superfície, os quais contribuíram para o consequente aumento de energia de superfície. No entanto, quando a hidrólise foi realizada na presença de PFDTEOS, obtiveram-se materiais híbridos com elevada hidrofobicidade e lipofobicidade (ângulos de contacto com água e diiodometano de 140º e 134º, respectivamente), devido à combinação da presença de grupos perfluorados e micro- e nano-rugosidades na superfície das fibras de celulose, conforme confirmado por SEM. Finalmente, a última abordagem permitiu preparar materiais derivados de celulose altamente hidrofóbicos e lipofóbicos (ângulos de contacto com água e diiodometano de 136º e 109º, respectivamente) por um processo simples, envolvendo tempos de tratamento tão curtos como 0.5 min. Este comportamento omnifóbico foi gerado pelo efeito sinergético entre a diminuição de energia de superfície das fibras, devido à presença de grupos metilo dos resíduos de TCMS ligados a estas, e a condensação dos resíduos de TCMS na forma de micro- e nano-partículas inorgânicas, que levou à criação de um revestimento rugoso à superfície das fibras, conforme observado por RMN de 29Si e SEM, respectivamente. A pré-humidificação das fibras de celulose demonstrou desempenhar um importante papel de “acelerador” dos processos de hidrólise e condensação das moléculas de TCMS. Nestas condições, o tempo de tratamento foi um dos parâmetros mais relevantes, pois para tempos de tratamento muito curtos (0.5 min) os materiais resultantes não apresentaram quaisquer diferenças a nível de propriedades físico-químicas em relação ao substrato de partida (a humidade em excesso consumiu todo o TCMS antes que este conseguisse reagir com os grupos hidroxilo das fibras de celulose), possuindo, por exemplo, valores de ângulos de contacto com água idênticos. Para tempos de tratamento mais longos, como 30 min, os materiais finais apresentaram a maior quantidade de componentes inorgânicos, tal como verificado por EA e TGA. Assim, o controlo da humidade das fibras é imperativo para se poder moldar as propriedades finais dos produtos. Esta última abordagem é particularmente promissora uma vez que tem como base um sistema simples e “verde” que pode ser facilmente implementado. Em conclusão, este trabalho permitiu demonstrar que a modificação química heterogénea controlada das fibras de celulose representa uma iniciativa promissora para a preparação de novos materiais obtidos a partir de recursos renováveis, com propriedades interessantes e passíveis de ser potencialmente aplicados em diferentes áreas. Para além do mais, as estratégias de modificação estudadas podem também ser precursoras de novos estudos que possam vir a ser desenvolvidos dentro do mesmo âmbito.

Metabonomic studies to assess beer quality and stability

The work reported in this thesis aimed at applying the methodology known as metabonomics to the detailed study of a particular type of beer and its quality control, with basis on the use of multivariate analysis (MVA) to extract meaningful information from given analytical data sets. In Chapter 1, a detailed description of beer is given considering the brewing process, main characteristics and typical composition of beer, beer stability and the commonly used analytical techniques for beer analysis. The fundamentals of the analytical methods employed here, namely nuclear magnetic resonance (NMR) spectroscopy, gas-chromatography-mass spectrometry (GC-MS) and mid-infrared (MIR) spectroscopy, together with the description of the metabonomics methodology are described shortly in Chapter 2. In Chapter 3, the application of high resolution NMR to characterize the chemical composition of a lager beer is described. The 1H NMR spectrum obtained by direct analysis of beer show a high degree of complexity, confirming the great potential of NMR spectroscopy for the detection of a wide variety of families of compounds, in a single run. Spectral assignment was carried out by 2D NMR, resulting in the identification of about 40 compounds, including alcohols, amino acids, organic acids, nucleosides and sugars. In a second part of Chapter 3, the compositional variability of beer was assessed. For that purpose, metabonomics was applied to 1H NMR data (NMR/MVA) to evaluate beer variability between beers from the same brand (lager), produced nationally but differing in brewing site and date of production. Differences between brewing sites and/or dates were observed, reflecting compositional differences related to particular processing steps, including mashing, fermentation and maturation. Chapter 4 describes the quantification of organic acids in beer by NMR, using different quantitative methods: direct integration of NMR signals (vs. internal reference or vs. an external electronic reference, ERETIC method) and by quantitative statistical methods (using the partial least squares (PLS) regression) were developed and compared. PLS1 regression models were built using different quantitative methods as reference: capillary electrophoresis with direct and indirect detection and enzymatic essays. It was found that NMR integration results generally agree with those obtained by the best performance PLS models, although some overestimation for malic and pyruvic acids and an apparent underestimation for citric acid were observed. Finally, Chapter 5 describes metabonomic studies performed to better understand the forced aging (18 days, at 45 ºC) beer process. The aging process of lager beer was followed by i) NMR, ii) GC-MS, and iii) MIR spectroscopy. MVA methods of each analytical data set revealed clear separation between different aging days for both NMR and GC-MS data, enabling the identification of compounds closely related with the aging process: 5-hydroxymethylfurfural (5-HMF), organic acids, γ-amino butyric acid (GABA), proline and the ratio linear/branched dextrins (NMR domain) and 5-HMF, furfural, diethyl succinate and phenylacetaldehyde (known aging markers) and, for the first time, 2,3-dihydro-3,5-dihydroxy-6-methyl-4(H)-pyran-4-one xii (DDMP) and maltoxazine (by GC-MS domain). For MIR/MVA, no aging trend could be measured, the results reflecting the need of further experimental optimizations. Data correlation between NMR and GC-MS data was performed by outer product analysis (OPA) and statistical heterospectroscopy (SHY) methodologies, enabling the identification of further compounds (11 compounds, 5 of each are still unassigned) highly related with the aging process. Data correlation between sensory characteristics and NMR and GC-MS was also assessed through PLS1 regression models using the sensory response as reference. The results obtained showed good relationships between analytical data response and sensory response, particularly for the aromatic region of the NMR spectra and for GC-MS data (r > 0.89). However, the prediction power of all built PLS1 regression models was relatively low, possibly reflecting the low number of samples/tasters employed, an aspect to improve in future studies.

Catalytic routes to convert saccharides to furanic aldehydes

The conversion of plant biomass-derived carbohydrates (preferably non-edible) into added-value products is envisaged to be at the core of the future biorefineries. Carbohydrates are the most abundant natural organic polymers on Earth. This work deals with the chemical valorisation of plant biomass, focusing on the acid-catalysed conversion of carbohydrates (mono and polysaccharides) to furanic aldehydes, namely 2-furaldehyde (Fur) and 5-hydroxymethyl-2-furaldehyde (Hmf), which are valuable platform chemicals that have the potential to replace a variety of oil derived chemicals and materials. The investigated reaction systems can be divided into two types depending on the solvent used to dissolve the carbohydrates in the reaction medium: water or ionic liquid-based systems. The reaction temperatures were greater than 150 ºC when the solvent was water, and lower than 150 º C in the cases of the ionic liquid-based catalytic systems. As alternatives to liquid acids (typically used in the industrial production of Fur), solid acid catalysts were investigated in these reaction systems. Aiming at the identification of (soluble and insoluble) reaction products, complementary characterisation techniques were used namely, FT-IR spectroscopy, liquid and solid state NMR spectroscopy, TGA, DSC and GC´GC-ToFMS analyses. Complex mixtures of soluble reaction products were obtained and different types of side reactions may occur. The requirements to be put on the catalysts for these reaction systems partly depend on the type of carbohydrates to be converted and the reaction conditions used. The thermal stability is important due to the fact that formation of humins and catalyst coking phenomena are characteristically inherent to these types of reactions systems leading to the need to regenerate the catalyst which can be effectively accomplished by calcination. Special attention was given to fully inorganic nanoporous solid acids, amorphous or crystalline, and consisting of nano to micro-size particles. The investigated catalysts were silicoaluminophosphates, aluminosilicates and zirconium-tungsten mixed oxides which are versatile catalysts in that their physicochemical properties can be fine-tuned to improve the catalytic performances in the conversion of different substrates (e.g. introduction of mesoporosity and modification of the acid properties). The catalytic systems consisting of aluminosilicates as solid acids and water as solvent seem to be more effective in converting pentoses and related polysaccharides into Fur, than hexoses and related polysaccharides into Hmf. The investigated solid acids exhibited fairly good hydrothermal stabilities. On the other hand, ionic liquid-based catalytic systems can allow reaching simultaneously high Fur and Hmf yields, particularly when Hmf is obtained from D-fructose and related polysaccharides; however, catalyst deactivation occurs and the catalytic reactions take place in homogeneous phase. As pointed out in a review of the state of the art on this topic, the development of truly heterogeneous ionic liquid-based catalytic systems for producing Fur and Hmf in high yields remains a challenge.

A RG-II type polysaccharide purified from Aconitum coreanum and their anti-inflammatory activity

Korean mondshood root polysaccharides (KMPS) isolated from the root of Aconitum coreanum (Lévl.) Rapaics have shown anti-inflammatory activity, which is strongly influenced by their chemical structures and chain conformations. However, the mechanisms of the anti-inflammatory effect by these polysaccharides have yet to be elucidated. A RG-II polysaccharide (KMPS-2E, Mw 84.8 kDa) was isolated from KMPS and its chemical structure was characterized by FT-IR and NMR spectroscopy, gas chromatography–mass spectrometry and high-performance liquid chromatography. The backbone of KMPS-2E consisted of units of [→6) -β-D-Galp (1→3)-β-L-Rhap-(1→4)-β-D-GalpA-(1→3)-β-D-Galp-(1→] with the side chain →5)-β-D-Arap (1→3, 5)-β-D-Arap (1→ attached to the backbone through O-4 of (1→3,4)-L-Rhap. T-β-D-Galp is attached to the backbone through O-6 of (1→3,6)-β-D-Galp residues and T-β-D-Ara is connected to the end group of each chain. The anti-inflammatory effects of KMPS-2E and the underlying mechanisms using lipopolysaccharide (LPS) - stimulated RAW 264.7 macrophages and carrageenan-induced hind paw edema were investigated. KMPS-2E (50, 100 and 200 µg/mL) inhibits iNOS, TLR4, phospho-NF-κB–p65 expression, phosphor-IKK, phosphor-IκB-α expression as well as the degradation of IκB-α and the gene expression of inflammatory cytokines (TNF-α, IL-1β, iNOS and IL-6) mediated by the NF-κB signal pathways in macrophages. KMPS-2E also inhibited LPS-induced activation of NF-κB as assayed by electrophorectic mobility shift assay (EMSA) in a dose-dependent manner and it reduced NF-κB DNA binding affinity by 62.1% at 200µg/mL. In rats, KMPS-2E (200 mg/kg) can significantly inhibit carrageenan-induced paw edema as ibuprofen (200 mg/kg) within 3 h after a single oral dose. The results indicate that KMPS-2E is a promising herb-derived drug against acute inflammation.

A pyrophosphate-responsive gadolinium(III) MRI contrast agent

This study shows that the relaxivity and optical properties of functionalised lanthanide-DTPA-bis-amide complexes (lanthanide=Gd3+ and Eu3+, DTPA=diethylene triamine pentaacetic acid) can be successfully modulated by addition of specific anions, without direct Ln3+/anion coordination. Zinc(II)-dipicolylamine moieties, which are known to bind strongly to phosphates, were introduced in the amide “arms” of these ligands, and the interaction of the resulting Gd–Zn2 complexes with a range of anions was screened by using indicator displacement assays (IDAs). Considerable selectivity for polyphosphorylated species (such as pyrophosphate and adenosine-5′-triphosphate (ATP)) over a range of other anions (including monophosphorylated anions) was apparent. In addition, we show that pyrophosphate modulates the relaxivity of the gadolinium(III) complex, this modulation being sufficiently large to be observed in imaging experiments. To establish the binding mode of the pyrophosphate and gain insight into the origin of the relaxometric modulation, a series of studies including UV/Vis and emission spectroscopy, luminescence lifetime measurements in H2O and D2O, 17O and 31P NMR spectroscopy and nuclear magnetic resonance dispersion (NMRD) studies were carried out.

DNA interaction and cytotoxicity studies of new ruthenium(II) cyclopentadienyl derivative complexescontaining heteroaromatic ligands

Four ruthenium(II) complexes with the formula [Ru(eta(5)-C(5)H(5))(PP)L][CF(3)SO(3)], being (PP = two triphenylphosphine molecules), L = 1-benzylimidazole, 1; (PP = two triphenylphosphine molecules), L = 2,2'bipyridine, 2; (PP = two triphenylphosphine molecules), L = 4-Methylpyridine, 3; (PP = 1,2-bis(diphenylphosphine) ethane), L = 4-Methylpyridine, 4, were prepared, in view to evaluate their potentialities as antitumor agents. The compounds were completely characterized by NMR spectroscopy and their crystal and molecular structures were determined by X-ray diffraction. Electrochemical studies were carried out giving for all the compounds quasi-reversible processes. The images obtained by atomic force microscopy (AFM) suggest interaction with pBR322 plasmid DNA. Measurements of the viscosity of solutions of free DNA and DNA incubated with different concentrations of the compounds confirmed this interaction. The cytotoxicity of compounds 1234 was much higher than that of cisplatin against human leukemia cancer cells (HL-60 cells). IC(50) values for all the compounds are in the range of submicromolar amounts. Apoptotic death percentage was also studied resulting similar than that of cisplatin. (C) 2010 Elsevier Inc. All rights reserved.

Development and application of NMR techniques to study water-in-CO2 microemulsions

Self-assembly is a phenomenon that occurs frequently throughout the universe. In this work, two self-assembling systems were studied: the formation of reverse micelles in isooctane and in supercritical CO2 (scCO2), and the formation of gels in organic solvents. The goal was the physicochemical study of these systems and the development of an NMR methodology to study them. In this work, AOT was used as a model molecule both to comprehensively study a widely researched system water/AOT/isooctane at different water concentrations and to assess its aggregation in supercritical carbon dioxide at different pressures. In order to do so an NMR methodology was devised, in which it was possible to accurately determine hydrodynamic radius of the micelle (in agreement with DLS measurements) using diffusion ordered spectroscopy (DOSY), the micellar stability and its dynamics. This was mostly assessed by 1H NMR relaxation studies, which allowed to determine correlation times and size of correlating water molecules, which are in agreement with the size of the shell that interacts with the micellar layer. The encapsulation of differently-sized carbohydrates was also studied and allowed to understand the dynamics and stability of the aggregates in such conditions. A W/CO2 microemulsion was prepared using AOT and water in scCO2, with ethanol as cosurfactant. The behaviour of the components of the system at different pressures was assessed and it is likely that above 130 bar reverse microemulsions were achieved. The homogeneity of the system was also determined by NMR. The formation of the gel network by two small molecular organogelators in toluene-d8 was studied by DOSY. A methodology using One-shot DOSY to perform the spectra was designed and applied with success. This yielded an understanding about the role of the solvent and gelator in the aggregation process, as an estimation of the time of gelation.

Production and characterization of Chitin-Glucan Complex by Komagataella pastoris

This thesis was focused on the production, extraction and characterization of chitin:β-glucan complex (CGC). In this process, glycerol byproduct from the biodiesel industry was used as carbon source. The selected CGC producing yeast was Komagataella pastoris (formerly known as Pichia pastoris), due the fact that to achieved high cell densities using as carbon source glycerol from the biodiesel industry. Firstly, a screening of K. pastoris strains was performed in shake flask assays, in order to select the strain of K. pastoris with better performance, in terms of growth, using glycerol as a carbon source. K. pastoris strain DSM 70877 achieved higher final cell densities (92-97 g/l), using pure glycerol (99%, w/v) and in glycerol from the biodiesel industry (86%, w/v), respectively, compared to DSM 70382 strain (74-82 g/l). Based on these shake flask assays results, the wild type DSM 70877 strain was selected to proceed for cultivation in a 2 l bioreactor, using glycerol byproduct (40 g/l), as sole carbon source. Biomass production by K. pastoris was performed under controlled temperature and pH (30.0 ºC and 5.0, respectively). More than 100 g/l biomass was obtained in less than 48 h. The yield of biomass on a glycerol basis was 0.55 g/g during the batch phase and 0.63 g/g during the fed-batch phase. In order to optimize the downstream process, by increasing extraction and purification efficiency of CGC from K. pastoris biomass, several assays were performed. It was found that extraction with 5 M NaOH at 65 ºC, during 2 hours, associated to neutralization with HCl, followed by successive washing steps with deionised water until conductivity of ≤20μS/cm, increased CGC purity. The obtained copolymer, CGCpure, had a chitin:glucan molar ratio of 25:75 mol% close to commercial CGC samples extracted from A. niger mycelium, kiOsmetine from Kitozyme (30:70 mol%). CGCpure was characterized by solid-state Nuclear Magnetic Resonance (NMR) spectroscopy and Differential Scanning Calorimetry (DCS), revealing a CGC with higher purity than a CGC commercial (kiOsmetine). In order to optimize CGC production, a set of batch cultivation experiments was performed to evaluate the effect of pH (3.5–6.5) and temperature (20–40 ºC) on the specific cell growth rate, CGC production and polymer composition. Statistical tools (response surface methodology and central composite design) were used. The CGC content in the biomass and the volumetric productivity (rp) were not significantly affected within the tested pH and temperature ranges. In contrast, the effect of pH and temperature on the CGC molar ratio was more pronounced. The highest chitin: β-glucan molar ratio (> 14:86) was obtained for the mid-range pH (4.5-5.8) and temperatures (26–33 ºC). The ability of K. pastoris to synthesize CGC with different molar ratios as a function of pH and temperature is a feature that can be exploited to obtain tailored polymer compositions.(...)

Nuclear magnetic resonance studies of boron trihalide complexes of 1,1-BIS (dimethylamino) ethylene and related bases

Boron tribalide complexes of 1,1-bis(dimethylamino)ethylene (DME) , t etramethylurea (TMU), tetramethylguanidine (TMG) , and pentamethylguanidine (PMG) and also mixed boron t r ihalide adducts of DME have been investigated by 1H and 19F NMR spectroscopy. Both nitrogen and the C-Q-H carbon of DME are possible donor a toms to boron trihal ides but complexation has been found to occur only at carbon of DME. The initial adduct acts as a Bronsted acid and gives up a proton to free DME in solut ion. A side reaction in the DME-BF, system gives rise to trace amounts of a complex aSSigned as (DME)2BF2+. (DME)2BF2+ is produced in much larger quantities in t he DME-BF3-BC13 and DME-BF,-BBr, systems by reaction of free DME with DME:BF2X (X = Cl, Br). Restricted r otation about the C-N bonds of TMUlBC13 and n1U:BBr3 has been observed at low temperatures. This complements previous work in this system and confirms oxygen donation of TMU to boron trihalides . Restricted rotation at low temperatures also has been observed in DMEboron trihalide systems

Half-sandwich silane complexes of ruthenium and iron : synthesis, structure and application to catalysis

The present thesis describes syntheses, structural studies, and catalytic reactivity of new non-classical silane complexes of ruthenium and iron. The ruthenium complexes CpRu(PPri3)CI(T]2-HSiR3) (1) (SiR3 = SiCh (a), SiClzMe (b), SiCIMe2 (c), SiH2Ph (d), SiMe2Ph (e» were prepared by reactions of the new unsaturated complex CpRu(PPri3)CI with silanes. According to NMR studies and X-ray analyses, the complexes la-c exhibit unusual simultaneous Si··· H and Si··· CI-Ru interactions. The complex CpRu(PPri3)CI was also used for the preparation of the first examples of late transition metal agostic silylamido complexes CpRu(PPri3)(N(T]2-HSiMe2)R) (2) (R= Ar or But), which were characterized by NMR spectroscopy. The iron complexes CpFe(PMePri2)H2(SiR3) (3) (SiR3 = SiCh (a), SiClzMe (b), SiCIMe2 (c), SiH2Ph (d), SiMe2Ph (e» were synthesized by the reaction of the new borohydride iron complex CpFe(PMePri2)(B~) with silanes in the presence NEt3. The complexes 3 exhibit unprecedented two simultaneous and equivalent Si··· H interactions, which was confirmed by X-ray analyses and DFT calculations. A series of cationic ruthenium complexes [CpRu(PR3)(CH3CN)(112-HSiR'3)]BAF (PR3 = PPri 3 (4), PPh3 (5); SiR'3 = SiCh (a), SiClzMe (b), SiClMe2 (c), SiH2Ph (d), SiMe2Ph (e» was obtained by substitution of one of the labile acetonitrile ligands in [CpRu(PR3)(CH3CNh]BAF with sHanes. Analogous complexes [TpRu(PR3)(CH3CN)(T]2 -HSiR' 3)]BAF (5) were obtained by the reaction of TpRu(PR3)(CH3CN)CI with LiBAF in the presence of silanes. The complexes 4-5 were characterized by NMR spectroscopy, and the observed coupling constants J(Si-H) allowed us to estimate the extent of Si-H bond activation in these compounds. The catalytic activity in hydrosilylation reactions of all of the above complexes was examined. The most promising results were achieved with the cationic ruthenium precatalyst [CpRu(PPri3)(CH3CN)2t (6). Complex 6 shows good to excellent catalytic activity in the hydrosilylation of carbonyls, dehydrogenative coupling of silanes with alcohols, amines, acids, and reduction of acid chlorides. We also discovered very selective reduction of nitriles and pyridines into the corresponding N-silyl imines and l,4-dihydropyridines, respectively, at room temperature with the possibility of catalyst recycling. These chemoselective catalytic methods have no analogues in the literature. The reactions were proposed to proceed via an ionic mechanism with intermediate formation of the silane a-complexes 4.

(A) BODIPY as a versatile fluorophore (B) Synthesis of PNAs via Staudinger reaction

(A) In recent years, 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) fluorophores have attracted considerable interest due to their unique photochemical properties. However detailed studies on the stability of BODIPY and analogues under acidic and basic conditions have been lacking. Thus the stability of a series of BODIPY analogues in acidic (di- and trichloroacetic acid) and basic (aqueous ammonium hydroxide) conditions was investigated using 11B NMR spectroscopy. Among the analogues tested, 4,4-diphenyl BODIPY was the most stable under the conditions used in the experiments. It was found that reaction of 4,4-dimethoxy BODIPY with dichloroacetic acid gave mixed anhydride 4,4-bis(dichloroacetoxy) BODIPY in good yields. Treatment of the latter mixed anhydride with alcohols such as methanol and ethanol in the presence of a base afforded corresponding borate esters, whereas treatment with 1,2-diols such as ethylene glycol and catechol in the presence of a base gave corresponding cyclic borate esters. Furthermore treatment of 4,4-difluoro-8-methyl-BODIPY with secondary amines in dihalomethane resulted in carbon–carbon bond formation at the meso-methyl position of BODIPY via Mannich-type reactions. The resulting modified BODIPY fluorophores possess high fluorescent quantum yields. Five BODIPY analogues bearing potential ion-binding moieties were synthesized via this Mannich-type reaction. Among these, the BODIPY bearing an aza-18-crown-5 tether was found to be selective towards copper (II) ion, resulting in a large blue shift in absorption and sharp fluorescent quenching, whereas aza-15-crown-4 analogue was selected towards fluoride ion, leading to effective florescent quenching and blue shift. (B) Peptide nucleic acids (PNA), as mimics of natural nucleic acids, have been widely applied in molecular biology and biotechnology. Currently, the preparation of PNA oligomers is commonly achieved by a coupling reaction between carboxyl and amino groups in the presence of an activator. In this thesis attempts were made towards the synthesis of PNA through the Staudinger ligation reactions between C-terminal diphenylphosphinomethanethiol thioesters and N-terminal α-azido PNA building blocks.

Études Structurales par Résonance Magnétique Nucléaire (RMN) du Site Actif du Ribozyme VS de Neurospora.

Nous étudions le ribozyme VS de Neurospora, en tant que système modèle, pour augmenter nos connaissances sur la relation entre la structure et la fonction chez les ARNs, ainsi que pour mieux comprendre le mécanisme de clivage de ce ribozyme. Il a été proposé précédemment que la boucle interne A730 dans la tige-boucle VI (SLVI) contient le site actif du ribozyme et lie un ou plusieurs ions métalliques qui pourraient participer au mécanisme réactionnel. Nous avons déterminé par spectroscopie RMN la structure de la tige-boucle SLVI contenant la boucle A730 afin d’éclaircir ce mécanisme. La structure obtenue est en accord avec les études biochimiques antérieures et présente un ou plusieurs sites de liaison au magnésium associé à la boucle interne. Suite à des études de cinétique et de mutagenèse, il a été proposé qu’une adénine localisée dans le site actif, A756, participe à la catalyse par acide/base générale. Des études de pH effectuées précédemment ont identifié un pKa catalytique (5.2-5.8) qui correspond probablement à l’équilibre de protonation du A756. À l’aide de méthodes utilisant le carbone-13, nous avons identifié un pKa modifié appartenant au A756, ce qui supporte le rôle de ce résidu dans la catalyse par acide/base générale. Les études structurales présentées ici aident donc à augmenter notre compréhension du mécanisme de clivage chez le ribozyme VS.

POCN-type Pincer Complexes of NiII and NiIII : synthesis, reactivities, catalytic activities and physical properties

Cette thèse décrit la synthèse, la caractérisation, les réactivités, et les propriétés physiques de complexes divalents et trivalents de Ni formés à partir de nouveaux ligands «pincer» de type POCN. Les ligands POCN de type amine sont préparés d’une façon simple et efficace via l’amination réductrice de 3-hydroxybenzaldéhyde avec NaBH4 et plusieurs amines, suivie par la phosphination de l’amino alcool résultant pour installer la fonction phosphinite (OPR2); le ligand POCN de type imine 1,3-(i-Pr)2PC6H4C(H)=N(CH2Ph) est préparé de façon similaire en faisant usage de PhCH2NH2 en l’absence de NaBH4. La réaction de ces ligands «pincer» de type POCN avec NiBr2(CH3CN)x en présence d’une base résulte en un bon rendement de la cyclométalation du lien C-H situé en ortho aux fonctions amine et phosphinite. Il fut découvert que la base est essentielle pour la propreté et le haut rendement de la formation des complexes «pincer» désirés. Nous avons préparé des complexes «pincer» plan- carrés de type POCN, (POCNRR΄)NiBr, possédant des fonctions amines secondaires et tertiaires qui démontrent des réactivités différentes selon les substituants R et R΄. Par exemple, les complexes possédant des fonctions amines tertiaires ArCH2NR2 (NR2= NMe2, NEt2, and morpholinyl) démontrent des propriétés rédox intéressantes et pourraient être convertis en leurs analogues trivalents (POCNR2)NiBr2 lorsque réagis avec Br2 ou N-bromosuccinimide (NBS). Les complexes trivalents paramagnétiques à 17 électrons adoptent une géométrie de type plan-carré déformée, les atomes de Br occupant les positions axiale et équatoriale. Les analyses «DSC» et «TGA» des ces composés ont démontré qu’ils sont thermiquement stables jusqu’à ~170 °C; tandis que la spectroscopie d’absorption en solution a démontré qu’ils se décomposent thermiquement à beaucoup plus basse température pour regénérer les complexes divalents ne possédant qu’un seul Br; l’encombrement stérique des substitutants amines accélère cette route de décomposition de façon significative. Les analogues NMe2 et N(morpholinyl) de ces espèces de NiIII sont actifs pour catalyser la réaction d’addition de Kharasch, de CX4 à des oléfines telles que le styrène, tandis qu’il fut découvert que l’analogue le moins thermiquement stable (POCNEt2)Ni est complètement inerte pour catalyser cette réaction. Les complexes (POCNRH)NiBr possédant des fonctions amines secondaires permettent l’accès à des fonctions amines substituées de façon non symétrique via leur réaction avec des halogénures d’alkyle. Un autre avantage important de ces complexes réside dans la possibilité de déprotonation pour préparer des complexes POCN de type amide. De telles tentatives pour déprotoner les fonctions NRH nous ont permis de préparer des espèces dimériques possédant des ligands amides pontants. La nature dimérique des ces complexes [P,C,N,N-(2,6-(i-Pr)2PC6H3CH2NR)Ni]2 (R= PhCH2 et Ph) fut établie par des études de diffraction des rayons-X qui ont démontré différentes géométries pour les cœurs Ni2N2 selon le substituant N : l’analogue (PhCH2)N possède une orientation syn des substitutants benzyles et un arrangement ressemblant à celui du cyclobutane du Ni et des atomes d’azote, tandis que l’analogue PhN adopte un arrangement de type diamant quasi-planaire des atomes du Ni et des atomes d’azote et une orientation anti des substituants phényles. Les espèces dimériques ne se dissocient pas en présence d’alcools, mais elles promouvoient l’alcoolyse catalytique de l’acrylonitrile. De façon intéressante, les rendements de ces réactions sont plus élevés avec les alcools possédant des fonctions O-H plus acides, avec un nombre de «turnover» catalytique pouvant atteindre 2000 dans le cas de m-cresol. Nous croyons que ces réactions d’alcoolyse procèdent par activation hétérolytique de l’alcool par l’espèce dimérique via des liaisons hydrogènes avec une ou deux des fonctions amides du dimère. Les espèces dimériques de Ni (II) s’oxydent facilement électrochimiquement et par reaction avec NBS ou Br2. De façon surprenante, l’oxydation chimique mène à l’isolation de nouveaux produits monomériques dans lesquels le centre métallique et le ligand sont oxydés. Le mécanisme d’oxydation fut aussi investigué par RMN, «UV-vis-NIR», «DFT» et spectroélectrochimie.

Complexes pinceurs de type POCOP de Nickel (II) : synthèse, caractérisation, réactivité et applications catalytiques

Ce mémoire décrit la synthèse, la caractérisation spectroscopique et l’étude de la réactivité catalytique d’une nouvelle série de complexes pinceurs de Ni(II) formés à partir du ligand POCOPPh (P,C,P-2,6-{Ph2PO}2C6H4), très peu étudié dans le cas du nickel. Les études décrites dans ce mémoire examinent l’effet des substituants des phosphines sur les propriétés spectroscopiques et électrochimiques ainsi que les activités catalytiques. La synthèse du ligand a été améliorée par rapport à la procédure connue dans la littérature en diminuant le temps de réaction à 30 min et la température jusqu'à température ambiante. Les composés pinceur (P,C,P-2,6-{Ph2PO}2C6H3)NiX ont été obtenus avec des rendements variant entre 60% et 88%. Le premier complexe a été synthétisé en faisant réagir le précurseur NiBr2(NCCH3)x avec le ligand POCOPPh pour donner (POCOPPh)NiBr. Ce dernier réagit par la suite avec les sels d’argent et de potassium pour donner 4 nouveaux complexes soient : (POCOPPh)NiCN, (POCOPPh)NiOTf, (POCOPPh)NiOAc et (POCOPPh)NiONO2 (OTf = triflate et OAc = acetate). Vu la réactivité limitée du dérivé bromure, le dérivé (POCOPPh)NiOTf a été utilisé pour la préparation du composé (POCOPPh)NiCCPh. Le dérivé Ni-OTf a été utilisé également pour la synthèse des complexes (POCOPPh)NiR qui ont été détectés par RMN. Ces complexes (POCOPPh)NiR ont montré une stabilité trop faible et donnent des nouveaux complexes de type (POCOPPh)NiX en échangeant l’halogène avec le Mg ou de type (POCOPPh)NiOH en s’hydrolysant. Les espèces cationiques [(POCOPPh)NiNCR][OTf] (R= Me, CHCH2, CHCHMe, C(Me)CH2, NCCH2CH2N(Ph)H) ont été obtenues facilement et avec des bon rendements à partir du (POCOPPh)NiOTf. Tous les composés obtenus ont été caractérisés par la spectroscopie RMN (1H, 13C{1H}, 31P{1H}, 19F{1H}), la spectroscopie IR et la spectroscopie UV-vis. L’analyse élémentaire et l’analyse par la diffraction des rayons X, dont le but est de résoudre la structure à l’état solide, ont été utilisées pour la plupart des complexes. Des études de voltampérométrie cyclique ont été menées pour déterminer la densité électronique des centres métalliques et l’effet des phosphines sur cette propriété électrochimique. Dans le but de déterminer l’effet des substituants des phosphines sur l’activité catalytique des complexes, nous avons évalué les réactivités catalytiques des deux complexes (POCOPPh)NiOTf et (POCOPi-Pr)NiOTf dans la réaction d’hydroamination des oléfines activés et plus spécifiquement l’acrylonitrile. Après optimisation des conditions expérimentales, on a constaté que la réactivité des deux composés sont similaires mais une grande différence apparaît après l’ajout des additifs. En effet, le complexe (POCOPi-Pr)NiOTf donne une bonne activité catalytique en présence de la triéthylamine, tandis que cette activité diminue considérablement en présence d’eau, contrairement au complexe (POCOPPh)NiOTf qui est plus actif en présence d’eau. Dans le cas du complexe (POCOPPh)NiOTf, on a pu montrer que la base se coordonne au nickel dans le produit formé après la réaction d’hydroamination, ce qui diminue l’activité de ce complexe dans certains cas. Également on a exploré la réaction de l’addition du lien O-H sur l’acrylonitrile, et étonnamment le complexe (POCOPPh)NiOTf est beaucoup plus actif que son homologue (POCOPi-Pr)NiOTf dans le cas des alcools aromatiques. Par contre, les alcools aliphatiques restent un défi majeur pour ce genre de complexe. Le mécanisme de cette réaction qui a été proposé montre que l’alcoolyse passe par les deux intermédiaires (POCOPPh)NiOAr et [(POCOPPh)NiOAr][HOAr] mais l’isolation de ces intermédiaires observés par RMN semble être difficile.

Méthodologie pour la synthèse combinatoire d’azapeptides: application à la synthèse d’analogues aza-GHRP-6 en tant que ligands du récepteur CD36

Les azapeptides sont des mimes peptidiques où le carbone alpha d’un ou de plusieurs acides aminés est remplacé par un atome d’azote. Cette modification tend à stabiliser une conformation en repliement beta en raison de la répulsion électronique entre les paires d’électrons libres des atomes d’azote adjacents et de la géométrie plane de l’urée. De plus, le résidu semicarbazide a une meilleure résistance face aux protéases en plus d’être chimiquement plus stable qu’une liaison amide. Bien que les propriétés des azapeptides en fassent des mimes peptidiques intéressants, leurs méthodes de synthèses font appel à la synthèse laborieuse d’hydrazines substituées en solution. Le peptide sécréteur d’hormone de croissance 6 (GHRP-6, His-D-Trp-Ala-Trp-D-Phe-Lys-NH2) est un hexapeptide synthétique qui possède une affinité pour deux récepteurs distincts: les récepteurs GHS-R1a et CD36. Les travaux effectués au cours de mon doctorat qui seront détaillés dans cet ouvrage visent à atteindre deux objectifs: (1) le développement d’analogues du peptide GHRP-6 sélectif à un seul récepteur et (2) la mise au point d’une nouvelle méthodologie pour la synthèse combinatoire d’azapeptides. En réponse au premier objectif, la synthèse parallèle de 49 analogues aza-GHRP-6 a été effectuée et certains candidats sélectifs au récepteur CD36 ont été identifiés. L’étude de leurs propriétés anti-angiogéniques, effectuée par nos collaborateurs, a également permis d’identifier des candidats intéressants pour le traitement potentiel de la dégénérescence maculaire liée à l’âge. Une nouvelle approche pour la synthèse combinatoire d’azapeptides, faisant appel à l’alkylation et la déprotection chimiosélective d’une sous-unité semicarbazone ancrée sur support solide, a ensuite été développée. La portée de cette méthodologie a été augmentée par la découverte de conditions permettant l’arylation régiosélective de cette sous-unité semicarbazone, donnant accès à treize nouveaux dérivés aza-GHRP-6 possédant des résidus aza-arylglycines aux positions D-Trp2 et Trp4. L’élaboration de conditions propices à l’alkylation et la déprotection chimiosélective de la semicarbazone a donné accès à une variété de chaînes latérales sur l’acide aminé « aza » préalablement inaccessibles. Nous avons, entre autres, démontré qu’une chaîne latérale propargyl pouvait être incorporée sur l’acide aminé « aza ». Tenant compte de la réactivité des alcynes, nous avons ensuite élaboré des conditions réactionnelles permettant la formation in situ d’azotures aromatiques, suivie d’une réaction de cycloaddition 1,3-dipolaire sur support solide, dans le but d’obtenir des mimes de tryptophane. Sept analogues du GHRP-6 ont été synthétisés et testés pour affinité au récepteur CD36 par nos collaborateurs. De plus, nous avons effectué une réaction de couplage en solution entre un dipeptide possédant un résidu aza-propargylglycine, du paraformaldehyde et une variété d’amines secondaires (couplage A3) afin d’accéder à des mimes rigides d’aza-lysine. Ces sous-unités ont ensuite été incorporées sur support solide afin de générer sept nouveaux azapeptides avec des dérivés aza-lysine à la position Trp4 du GHRP-6. Enfin, une réaction de cyclisation 5-exo-dig a été développée pour la synthèse de N-amino imidazolin-2-ones en tant que nouveaux mimes peptidiques. Leur fonctionnalisation par une série de groupements benzyliques à la position 4 de l’hétérocycle a été rendue possible grâce à un couplage Sonogashira précédant la réaction de cyclisation. Les propriétés conformationnelles de cette nouvelle famille de composés ont été étudiées par cristallographie aux rayons X et spectroscopie RMN d’un tétrapeptide modèle. L’activité biologique de deux mimes peptidiques, possédant un résidu N-amino-4-méthyl- et 4-benzyl-imidazolin-2-one à la position Trp4 du GHRP-6, a aussi été examinée. L’ensemble de ces travaux devrait contribuer à l’avancement des connaissances au niveau des facteurs structurels et conformationnels requis pour le développement d’azapeptides en tant que ligands du récepteur CD36. De plus, les résultats obtenus devraient encourager davantage l’utilisation d’azapeptides comme peptidomimétiques grâce à leur nouvelle facilité de synthèse et la diversité grandissante au niveau de la chaîne latérale des acides aminés « aza ».