Chiral thiouronium salts: synthesis, characterisation and application in NMR enantio-discrimination of chiral oxoanions

Chiral thioureas and functionalised chiral thiouronium salts were synthesised starting from the relatively cheap and easily available chiral amines: (S)-methylbenzylamine and rosin-derived (+)-dehydroabietylamine. The introduction of a delocalised positive charge to the thiourea functionality, by an alkylation reaction at the sulfur atom, enables dynamic rotameric processes: hindered rotations about the delocalised CN and CS bonds. Hence, four different rotamers/isomers may be recognised: syn-syn, syn-anti, anti-syn and anti-anti. Extensive H-1 and C-13 NMR studies have shown that in hydrogen-bond acceptor solvents, such as perdeuteriated dimethyl sulfoxide, the syn-syn conformation is preferable. On the other hand, when using non-polar solvents, such as CDCl3, the mixture of syn-syn and syn-anti isomers is detectable, with an excess of the latter. Apart from this, in the case of S-butyl-N,N'-bis(dehydroabietyl)thiouronium ethanoate in CDCl3, the H-1 NMR spectrum revealed that strong bifurcated hydrogen bonding between the anion and the cation causes global rigidity without signs of hindered rotamerism observable on the NMR time scale. This suggested that these new salts might be used as NMR discriminating agents for chiral oxoanions, and are indeed more effective than their archetypal guanidinium analogues or the neutral thioureas. The best results in recognition of a model substrate, mandelate, were obtained with S-butyl-N,N'-bis(dehydroabietyl) thiouronium bistriflamide. It was confirmed that the chiral recognition occurred not only for carboxylates but also for sulfonates and phosphonates. Further H-1 NMR studies confirmed a 1 : 1 recognition mode between the chiral agent (host) and the substrate (guest); binding constants were determined by H-1 NMR titrations in solutions of DMSO-d(6) in CDCl3. It was also found that the anion of the thiouronium salt had a significant influence on the recognition process: anions with poor hydrogen-bond acceptor abilities led to the best discrimination. The presence of host-guest hydrogen bonding was confirmed in the X-ray crystal structure of S-butyl-N,N'-bis(dehydroabietyl)thiouronium bromide and by computational studies (density functional theory).

Apolipoprotein C-III protein concentrations and gene polymorphisms in type 1 diabetes:associations with lipoprotein subclasses

Serum apolipoprotein C-III (apoCIII) concentration and apoCIII gene polymorphisms have been shown to be a risk factor for cardiovascular disease; however, the underlying mechanisms remain unclear. In addition, no studies have been performed that address these issues in type 1 diabetes. The current study investigated apoCIII protein and apoCIII gene variation in a normotriglyceridemic (82 +/- 57 mg/dL) population of patients with type 1 diabetes, the Diabetes Control and Complications Trial/Epidemiology of Diabetes Intervention and Complications (DCCT/EDIC) cohort. Blood samples were obtained in 409 patients after an overnight fast. Serum apoCIII concentration was highly correlated with multiple changes in lipids and lipoproteins that resulted in an adverse cardiovascular disease risk profile. Higher apoCIII concentrations were associated (P <.0001) with increased triglycerides (r = 0.78), total (r = 0.61) and low-density lipoprotein (LDL) (r = 0.40) cholesterol, apoA-I (r = 0.26), and apoB (r = 0.50), and these relationships persisted after controlling for age, gender, body mass index (BMI), and hemoglobin A1c (HbA1c). Nuclear magnetic resonance (NMR) lipoprotein subclass analyses demonstrated that apoCIII was correlated with an increase in very-low-density lipoprotein (VLDL) subclasses (P = .0001). There also was a highly significant positive relationship between serum apoCIII concentration and the LDL particle concentration in both men (r = 0.49, P = .001) and women (r = 0.40, P = .001), and a highly significant negative relationship between serum apoCIII levels and average LDL particle size in both men (r = -0.37, P = .001) and women (r = -0.22, P = .001) due primarily to an augmentation in the small L1 subclass (r = 0.42, P = .0001). Neither the T(-455) --> C polymorphism affecting an insulin response element in the apoCIII gene promoter nor a SacI polymorphism in the 3'UTR were associated with any alterations in circulating apoCIII concentrations, serum lipids, apolipoprotein concentrations, lipoprotein composition, or parameters measured by NMR lipoprotein subclass analyses. In summary, elevated apoCIII concentration was associated with risk factors for cardiovascular disease in normolipidemic type 1 diabetic patients through associated changes in lipoprotein subfraction distributions, which were independent of apoCIII genotype.

In vivo glycated low-density lipoprotein is not more susceptible to oxidation than nonglycated low-density lipoprotein in type 1 diabetes

It has been suggested that low-density lipoprotein (LDL) modified by glycation may be more susceptible to oxidation and thus, enhance its atherogenicity. Using affinity chromatography, LDL glycated in vivo (G-LDL) and relatively nonglycated. (N-LDL) subfractions can be isolated from the same individual. The extent of and susceptibility to oxidation of N-LDL compared with G-LDL was determined in 15 type 1 diabetic patients. Total LDL was isolated and separated by boronate affinity chromatography into relatively glycated (G-) and nonglycated (N-) subfractions. The extent of glycation, glycoxidation, and lipoxidation, lipid soluble antioxidant content, susceptibility to in vitro oxidation, and nuclear magnetic resonance (NMR)-determined particle size and subclass distribution were determined for each subfraction. Glycation, (fructose-lysine) was higher in G-LDL versus N-LDL, (0.28 +/- 0.08 v 0.13 +/- 0.04 mmol/mol lysine, P <.0001). However, levels of glycoxidation/lipoxidation products and of antioxidants were similar or lower in G-LDL compared with N-LDL and were inversely correlated with fructose-lysine (FL) concentrations in G-LDL, but positively correlated in N-LDL. In vitro LDL (CuCl2) oxidation demonstrated a longer lag time for oxidation of G-LDL than N-LDL (50 +/- 0.16 v 37 +/- 0.15 min, P <.01), but there was no difference in the rate or extent of lipid oxidation, nor in any aspect of protein oxidation. Mean LDL particle size and subclass distribution did not differ between G-LDL and N-LDL. Thus, G-LDL from well-controlled type 1 diabetic patients is not more modified by oxidation, more susceptible to oxidation, or smaller than relatively N-LDL, suggesting alternative factors may contribute to the atherogenicity of LDL from type 1 diabetic patients.

The phase behaviour of 1-alkyl-3-methylimidazolium tetrafluoroborates; ionic liquids and ionic liquid crystals

Air- and water-stable 1-alkyl-3-methylimidazolium tetrafluoroborate salts with the general formula [C-mim][BF] (n = 0-18) have been prepared by metathesis from the corresponding chloride or bromide salts. The salts have been characterised by H NMR and IR spectroscopy, microanalysis, polarising optical microscopy and differential scanning calorimetry. Those with short alkyl chains (n = 2-10) are isotropic ionic liquids at room temperature and exhibit a wide liquid range, whereas the longer chain analogues are low melting mesomorphic crystalline solids which display an enantiotropic smectic A mesophase. The thermal range of the mesophase increases with increasing chain length and in the case of the longest chain salt prepared, [C-mim][BF], the mesophase range is ca. 150°C.

Comparative study on transport properties for LiFAP and LiPF6 in alkyl-carbonates as electrolytes through conductivity, viscosity and NMR self-diffusion measurements

We present in this work a comparative study on density and transport properties, such as the conductivity (sigma), viscosity (eta) and self-diffusion coefficients (D), for electrolytes based on the lithium hexafluorophosphate, LiPF6; or on the lithium tris(pentafluoroethane)-trifluorophosphate, LiFAP dissolved in a binary mixture of ethylene carbonate (EC) and dimethylcarbonate (DMC) (50:50 wt%). For each electrolyte, the temperature dependence on transport properties over a temperature range from 10 to 80 degrees C and 20 to 70 degrees C for viscosity and conductivity, respectively, exhibits a non-Arrhenius behavior. However, this dependence is correctly correlated by using the Vogel-Tamman-Fulcher (VTF) type fitting equation. In each case, the best-fit parameters, such as the pseudo activation energy and ideal glass transition temperature were then extracted. The self-diffusion coefficients (D) of the Li+ cation and PF6- or FAP(-) anions species, in each studied electrolyte, were then independently determined by observing Li-3, F-19 and P-31 nuclei with the pulsed-gradient spin-echo (PGSE) NMR technique over the same temperature range from 20 to 80 degrees C. Results show that even if the diffusion of the lithium cation is quite similar in both electrolytes, the anions diffusion differs notably. In the case of the LiPF6-based electrolyte, for example at T approximate to 75 degrees C (high temperature), the self-diffusion coefficients of Li+ cations in solution (D (Li+)approximate to 5 x 10(-19) m(2) s(-1)) is 1.6 times smaller than that of PF6- anions (D (PF6-) = 8.5 x 10(-19) m(2) s(-1)), whereas in the case of the LiFAP-based electrolyte, FAP(-) anions diffuse at same rate as the Li+ cations (D (FAP(-)) = 5 x 10(-1) m(2) s(-1)). Based on these experimental results, the transport mobility of ions were then investigated through Stokes-Einstein and Nernst-Einstein equations to determine the transport number of lithium t(Li)(+), effective radius of solvated Li+ and of PF6- and FAP(-) anions, and the degree of dissociation of these lithium salts in the selected EC/DMC (50:50 wt%) mixture over a the temperature range from 20 to 80 degrees C. This study demonstrates the conflicting nature of the requirements and the advantage of the well-balanced properties as ionic mobility and dissociation constant of the selected electrolytes. (C) 2013 Elsevier Ltd. All rights reserved.

Tailoring ionic liquid catalysts: structure, acidity and catalytic activity of protonic ionic liquids based on anionic clusters, [(HSO4)(H2SO4)x]− (x = 0, 1, or 2)

Aiming at inexpensive Brønsted-acidic ionic liquids, suitable for industrial-scale catalysis, a family of protonic ionic liquids based on nitrogen bases and sulfuric acid has been developed. Variation of the molar ratio of sulfuric acid, χH2SO4, was used to tune acidity. The liquid structure was studied using 1H NMR and IR spectroscopies, revealing the existence of hydrogen-bonded clusters, [(HSO4)(H2SO4)]−, for χH2SO4 > 0.50. Acidity, quantified by Gutmann Acceptor Number (AN), was found to be closely related to the liquid structure. The ionic liquids were employed as acid catalysts in a model reaction; Fischer esterification of acetic acid with 1-butanol. The reaction rate depended on two factors; for χH2SO4 > 0.50, the key parameter was acidity (expressed as AN value), while for χH2SO4 > 0.50 it was the mass transport (solubility of starting materials in the ionic liquid phase). Building on this insight, the ionic liquid catalyst and reaction conditions have been chosen. Conversion values of over 95% were achieved under exceptionally mild conditions, and using an inexpensive ionic liquid, which could be recycled up to eight times without diminution in conversion or selectivity. It has been demonstrated how structural studies can underpin rational design and development of an ionic liquid catalyst, and in turn lead to a both greener and economically viable process.

Photocatalytic organic synthesis in an NMR tube:CC coupling of phenoxyacetic acid and acrylamide

NMR was used to study the semiconductor photocatalytic (SPC) CC coupling of phenoxyacetic acid (PAA) with acrylamide (ACM) in an NMR tube photoreactor. Using an NMR tube with a sol-gel titania inner coating as a photoreactor, this reaction is relatively clean, forming only 1 product, 4-phenoxybutanamide (4-PB), in yields up to 78%. This SPC reaction is used to assess the activity of the sol-gel titania coating as a function of their annealing temperature, which alters the surface area and phase of the titania, and the general reusability of the TiO coated NMR tubes. The optimum temperature range for annealing the sol-gel titania films is between 450 °C and 800 °C, with the maximum yield and rate attained at 450 °C. Despite a decrease in the initial rates of formation of 4-PB above an annealing temperature of 450 °C, the final product yields remained similar, giving maximum yields within 60 min of irradiation. The reusability study reveals that the activity of the sol-gel titania can quickly deteriorate with repeated use due to the adsorption of yellow/brown coloured, insoluble, most likely organic polymeric, material and its screening effect on the underlying photocatalyst. The titania can, however, be restored to its original activity by a simple heat treatment at 450 °C for 30 min.

Toluene dioxygenase-catalysed oxidation route to angular cis-monohydrodiols and other bioproducts from bacterial metabolism of 1,2-dihydrobenzocyclobutene and derivatives

A mutant strain (UV4) of the soil bacterium Pseudomonas putida, containing toluene dioxygenase, has been used in the metabolic oxidation of 1,2-dihydrobenzocyclobutene 12 dagger and the related substrates 1,2-dihydrobenzocyclobuten-1-ol 13 and biphenylene 33. Stable angular cis-monohydrodiol metabolites (1R,2S)-bicyclo[4.2.0]octa-3,5-diene-1,2 7, (1S,2S,8S)-bicyclo[4.2.0]octa-3,5-diene-1,2,8-triol 8 and biphenylene-cis-1,8b-diol 9, isolated from each of these substrates, have been structurally and stereochemically assigned. The structure, enantiopurity and absolute configuration of the other cis-diol metabolites, (2R,3S)-bicyclo[4.2.0]octa-1(6),4-diene-2,3-diol 14 and cis-1,2-dihydroxy-1,2-dihydrobenzocyclobutene 16, and the benzylic oxidation bioproducts, 1,2-dihydrobenzocyclobuten-1-ol 13, 1,2-dihydrobenzocyclobuten-1-one 15 and 2-hydroxy-1,2-dihydrobenzocyclobuten-1-one 17, obtained from 1,2-dihydrobenzocyclobutene and 1,2-dihydrobenzocyclobuten-1-ol, have been determined with the aid of chiral stationary-phase HPLC, NMR and CD spectroscopy, and stereochemical correlation. X-Ray crystallographic methods have been used in the determination of absolute configuration of the di-camphanates 27 (from diol 7) and 32 (from diol 9), and the di-MTPA ester 29 (from diol 14) of the corresponding cis-diol metabolites. The metabolic sequence involved in the formation of bioproducts derived from 1,2-dihydrobenzocyclobutene 12 has been investigated.

Data on carotid intima-media thickness and lipoprotein subclasses in type 1 diabetes from the Diabetes Control and Complications Trial and the Epidemiology of Diabetes Interventions and Complications (DCCT/EDIC)

Type 1 diabetes (T1DM) is associated with increased risk of macrovascular complications. We examined longitudinal associations of serum conventional lipids and nuclear magnetic resonance (NMR)-determined lipoprotein subclasses with carotid intima-media thickness (IMT) in adults with T1DM (n=455) enrolled in the Diabetes Control and Complications Trial (DCCT). Data on serum lipids and lipoproteins were collected at DCCT baseline (1983-89) and were correlated with common and internal carotid IMT determined by ultrasonography during the observational follow-up of the DCCT, the Epidemiology of Diabetes Interventions and Complications (EDIC) study, at EDIC 'Year 1' (199-1996) and EDIC 'Year 6' (1998-2000). This article contains data on the associations of DCCT baseline lipoprotein profiles (NMR-based VLDL & chylomicrons, IDL/LDL and HDL subclasses and 'conventional' total, LDL-, HDL-, non-HDL-cholesterol and triglycerides) with carotid IMT at EDIC Years 1 and 6, stratified by gender. The data are supplemental to our original research article describing detailed associations of DCCT baseline lipids and lipoprotein profiles with EDIC Year 12 carotid IMT (Basu et al. in press) [1].

Nuclear magnetic resonance-determined lipoprotein subclasses and carotid intima-media thickness in type 1 diabetes

BACKGROUND: Dyslipidemia has been linked to vascular complications of Type 1 diabetes (T1DM). We investigated the prospective associations of nuclear magnetic resonance-determined lipoprotein subclass profiles (NMR-LSP) and conventional lipid profiles with carotid intima-media thickness (IMT) in T1DM.

METHODS: NMR-LSP and conventional lipids were measured in a subset of Diabetes Control and Complications Trial (DCCT) participants (n = 455) at study entry ('baseline', 1983-89), and were related to carotid IMT determined by ultrasonography during the observational follow-up of the DCCT, the Epidemiology of Diabetes Interventions and Complications (EDIC) study, at EDIC Year 12 (2004-2006). Associations were defined using multiple linear regression stratified by gender, and following adjustment for HbA1c, diabetes duration, body mass index, albuminuria, DCCT randomization group, smoking status, statin use, and ultrasound devices.

RESULTS: In men, significant positive associations were observed between some baseline NMR-subclasses of LDL (total IDL/LDL and large LDL) and common and/or internal carotid IMT, and between conventional total- and LDL-cholesterol and non-HDL-cholesterol and common carotid IMT, at EDIC Year 12; these persisted in adjusted analyses (p < 0.05). Large LDL particles and conventional triglycerides were positively associated with common carotid IMT changes over 12 years (p < 0.05). Inverse associations of mean HDL diameter and large HDL concentrations, and positive associations of small LDL with common and/or internal carotid IMT (all p < 0.05) were found, but did not persist in adjusted analyses. No significant associations were observed in women.

CONCLUSION: NMR-LSP-derived LDL particles, in addition to conventional lipid profiles, may help in identifying men with T1DM at highest risk for vascular disease.

Síntese e transformações de compostos do tipo pirazol e 1,2,3-triazol

Compostos do tipo pirazol e 1,2,3-triazol encontram-se presentes em inúmeras moléculas biologicamente ativas. Muitos fármacos atualmente comercializados ou em fase de estudos clínicos contêm na sua estrutura base núcleos de pirazol ou 1,2,3-triazol. Por isso, estes compostos têm sido alvo de intensa pesquisa na procura de novas moléculas com potenciais aplicações medicinais e agroquímicas. Nesta dissertação são descritas novas vias de síntese de novos compostos do tipo pirazol e 1,2,3-triazol. No primeiro capítulo apresenta-se uma breve revisão bibliográfica sobre a atividade biológica, ocorrência natural e métodos de síntese de pirazóis e seus derivados. O segundo capítulo foca-se na síntese de (E)-2-estiril-3-halo-4H-cromen-4-onas e sua transformação em 3(5)-aril-5(3)-[2-(2-hidroxifenil)-2-oxoetil-1H-pirazóis. Em primeiro lugar faz-se uma revisão bibliográfica sobre as (E)-2-estiril-4H-cromen-4-onas e a sua semelhança estrutural com as flavonas, a sua importância e ocorrência natural e métodos de síntese. São ainda abordadas as metodologias mais utilizadas para a síntese de derivados halogenados de (E)-2-estiril-4H-cromen-4-onas. Seguidamente são apresentados e discutidos os resultados da síntese de (E)-3-bromo-2-estiril-4H-cromen-4-onas através da reação de 5-aril-3-hidroxi-1-(2-hidroxifenil)penta-2,4-dien-1-onas com NBS, sob irradiação com micro-ondas, tendo sido estabelecida uma nova metodologia mais eficiente, rápida e regiosseletiva para a síntese de (E)-3-bromo-2-estiril-4H-cromen-4-onas, na ausência de solvente. São igualmente apresentados os resultados da síntese regiosseletiva de (E)-2-estiril-3-iodo-4H-cromen-4-onas através da reação de 5-aril-3-hidroxi-1-(2-hidroxifenil)penta-2,4-dien-1-onas com NIS e TFA/TFAA/NaOAc. Em ambos os métodos de halogenação desenvolvidos, obtiveram-se como produtos secundários as (E)-2-estiril-4H-cromen-4-onas correspondentes. Seguidamente é apresentado o estudo da reação de (E)-2-estiril-3-halo-4H-cromen-4-onas com hidrato de hidrazina. Ao contrário do esperado, obtiveram-se os 3(5)-aril-5(3)-[2-(2-hidroxifenil)-2-oxoetil-1H-pirazóis através de uma reação de adição conjugada 1,6-, de hidrazina à posição C- da cromona com consequente abertura do anel, seguida de uma adição conjugada 1,4- intramolecular. Estes resultados demonstraram que esta reação segue um mecanismo diferente daquele que está reportado na literatura para a reação de (E)-2-estiril-4H-cromen-4-onas não halogenadas em C-3 com hidrato de hidrazina. No terceiro capítulo apresenta-se uma breve revisão bibliográfica sobre as propriedades, aplicações e metodologias de síntese de 1,2,3-triazóis, dando mais relevância às reações de cicloadição 1,3-dipolar e de “click-chemistry”. Seguidamente descrevem-se os resultados obtidos na reação de (E)-5(3)-estiril-3(5)-(2-hidroxifenil)-1H-pirazóis com a azida de sódio para obtenção de díades pirazol-1,2,3-triazol. No entanto esta reação deu origem a novos 5(3)-(2-aril-2-azidoetil)-3(5)-(2-hidroxifenil)-1H-pirazóis e não às díades pirazol-1,2,3-triazol pretendidas. Como o resultado não foi o esperado, desenvolveu-se outra metodologia de síntese, que envolve, num primeiro, a reação de (E)-2-estiril-4H-cromen-4-onas com azida de sódio, dando origem a 5(4)-aril-4(5)-(cromon-2-il)-1H-1,2,3-triazóis. No passo seguinte, efetuou-se a reação destes compostos com hidrato de hidrazina tendo ocorrido a formação das diades 5(4)-aril-4(5)-[3(5)-(2-hidroxifenil)-1H-pirazol-5(3)-il]-1H-1,2,3-triazol pretendidas. No quarto capítulo, estudou-se a reatividade de (E)-5(3)-estiril-3(5)-(2-hidroxifenil)-1H-pirazóis em reações de iodação com vista à obtenção de 4-iodo-1H-pirazóis. Apresenta-se uma breve revisão bibliográfica sobre os diferentes métodos descritos na literatura para a iodação de compostos heterocíclicos aromáticos, nomeadamente para a obtenção de 4-iodo-1H-pirazóis. Dos vários sistemas de iodação testados, o sistema oxidativo I2/CAN foi o que deu melhores resultados na iodação dos (E)-5(3)-estiril-3(5)-(2-hidroxifenil)-1H-pirazóis. Este método permitiu iodar a posição C-4 do núcleo de pirazol apenas para os derivados que possuem o grupo nitro ou o átomo de cloro no anel do grupo estirilo, obtendo-se o 3(5)-(2-hidroxifenil)-4-iodo-5(3)-(4-nitrofenil)vinil-1H-pirazol e o 5(3)-(4-clorofenil)vinil)-3(5)-(2-hidroxi-5-iodofenil)-4-iodo-1H-pirazol; no entanto, para os restantes derivados, verificou-se apenas a iodação nas posições ativadas do anel fenólico. Todos os novos compostos sintetizados foram caraterizados estruturalmente recorrendo a estudos de espetroscopia de ressonância magnética nuclear (RMN) mono e bidimensionais. Sempre que possível, para uma caraterização estrutural mais completa, foram efetuados espetros de massa (EM) e análises elementares ou espetros de massa de alta resolução (EMAR) para todos os novos compostos sintetizados. Finalmente são apresentadas as conclusões gerais deste trabalho e perspetivas futuras.

Evaluation of the matabolic response of osteosarcoma cells to conventional and new anticancer drugs by NMR metabolomics

The main scope of this work was to evaluate the metabolic effects of anticancer agents (three conventional and one new) in osteosarcoma (OS) cells and osteoblasts, by measuring alterations in the metabolic profile of cells by nuclear magnetic resonance (NMR) spectroscopy metabolomics. Chapter 1 gives a theoretical framework of this work, beginning with the main metabolic characteristics that globally describe cancer as well as the families and mechanisms of action of drugs used in chemotherapy. The drugs used nowadays to treat OS are also presented, together with the Palladium(II) complex with spermine, Pd2Spm, potentially active against cancer. Then, the global strategy for cell metabolomics is explained and the state of the art of metabolomic studies that analyze the effect of anticancer agents in cells is presented. In Chapter 2, the fundamentals of the analytical techniques used in this work, namely for biological assays, NMR spectroscopy and multivariate and statistical analysis of the results are described. A detailed description of the experimental procedures adopted throughout this work is given in Chapter 3. The biological and analytical reproducibility of the metabolic profile of MG-63 cells by high resolution magic angle spinning (HRMAS) NMR is evaluated in Chapter 4. The metabolic impact of several factors (cellular integrity, spinning rate, temperature, time and acquisition parameters) on the 1H HRMAS NMR spectral profile and quality is analysed, enabling the definition of the best acquisition parameters for further experiments. The metabolic consequences of increasing number of passages in MG-63 cells as well as the duration of storage are also investigated. Chapter 5 describes the metabolic impact of drugs conventionally used in OS chemotherapy, through NMR metabolomics studies of lysed cells and aqueous extracts analysis. The results show that MG-63 cells treated with cisplatin (cDDP) undergo a strong up-regulation of lipid contents, alterations in phospholipid constituents (choline compounds) and biomarkers of DNA degradation, all associated with cell death by apoptosis. Cells exposed to doxorubicin (DOX) or methotrexate (MTX) showed much slighter metabolic changes, without any relevant alteration in lipid contents. However, metabolic changes associated with altered Krebs cycle, oxidative stress and nucleotides metabolism were detected and were tentatively interpreted at the light of the known mechanisms of action of these drugs. The metabolic impact of the exposure of MG-63 cells and osteoblasts to cDDP and the Pd2Spm complex is described in Chapter 6. Results show that, despite the ability of the two agents to bind DNA, the metabolic consequences that arise from exposure to them are distinct, namely in what concerns to variation in lipid contents (absent for Pd2Spm). Apoptosis detection assays showed that, differently from what was seen for MG-63 cells treated with cDDP, the decreased number of living cells upon exposure to Pd2Spm was not due to cell death by apoptosis or necrosis. Moreover, the latter agent induces more marked alterations in osteoblasts than in cancer cells, while the opposite seemed to occur upon cDDP exposure. Nevertheless, the results from MG-63 cells exposure to combination regimens with cDDP- or Pd2Spm-based cocktails, described in Chapter 7, revealed that, in combination, the two agents induce similar metabolic responses, arising from synergy mechanisms between the tested drugs. Finally, the main conclusions of this thesis are summarized in Chapter 8, and future perspectives in the light of this work are presented.

Solid-state NMR and powder XRD studies of the structure of SAPO-40 upon hydration-dehydration cycles

It is well known that after the removal of the template many porous aluminophosphates and related materials are very sensitive to water.' Depending on the type of structure, reversible or irreversible phase transitions, loss of crystallinity and changes in the coordination of some framework A1 upon rehydration are observed. For example, solid-state NMR shows that the rehydration of SAPO-5 leads to the formation of octahedral Al. Subsequent dehydration restores the initial tetrahedral coordination of Al. Template-free SAPO-37 becomes totally amorphous to X-rays after exposure to water and stays so after subsequent thermal treatment^.,,^ In contrast, Barthomeuf and co-workers have shown recently, that, on hydration, template-free SAPO-34, an analogue of chabasite, shows the opening of some Si-0-A1 bonds, the effect being reversible upon dehydrati~n.T~h e hydrated distorted structure was found to be stable for months with no further modifications and the ordered material could be regenerated by removal of water. Here we wish to report that the structure of template-free SAPO-40 undergoes a similar reversible modification.

Simulation of the magnetic induction vector of a magnetic core to be used in FFC NMR relaxometry

This paper is a contribution for the assessment and comparison of magnet properties based on magnetic field characteristics particularly concerning the magnetic induction uniformity in the air gaps. For this aim, a solver was developed and implemented to determine the magnetic field of a magnetic core to be used in Fast Field Cycling (FFC) Nuclear Magnetic Resonance (NMR) relaxometry. The electromagnetic field computation is based on a 2D finite-element method (FEM) using both the scalar and the vector potential formulation. Results for the magnetic field lines and the magnetic induction vector in the air gap are presented. The target magnetic induction is 0.2 T, which is a typical requirement of the FFC NMR technique, which can be achieved with a magnetic core based on permanent magnets or coils. In addition, this application requires high magnetic induction uniformity. To achieve this goal, a solution including superconducting pieces is analyzed. Results are compared with a different FEM program.

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.