Bis(dimethylamino)-2-(4-methoxyphenyl)naphthalene (3) and 1,8-bis(dimethylamino)-4-diphenylphosphonaphthalene (5b) were synthesized as ESI-active analogues of the common organometallic ligands η6-anisole and triphenylphosphine. The water-soluble phosphine, sodium triphenylphosphine monosulfonate, was re-purposed as an ESI-active ligand. Its solubility in organic solvents and amenability to electrospray ionization was improved by replacing Na+ with the non-coordinating bis(triphenylphosphine)iminium cation. A new sample introduction method named PSI (pressurized sample infusion) was developed for the continuous infusion of air/moisture-sensitive samples into the mass spectrometer. The flow rate can be determined using a modified version of the Hagen-Poiseuille equation, and the ability of PSI (coupled with an ESI tag) to give quantitative kinetic data is demonstrated. A method for maintaining a dry, air-free ESI source is described for the analysis of highly reactive samples. The above developments were applied to the study of the copper-free Sonogashira (Heck alkynylation) reaction. The proposed active catalyst (Pd(0)L2, where L = PPh3 or 7) was observed, and its reactivity with iodomethane in the gas phase was determined to be less than that of Pd(0)L. Nevertheless, Pd(0)L2 is extremely reactive and even oxidatively adds dichloromethane (t1/2 = 10.7 min at 40 °C). Under standard reaction conditions intermediates corresponding to oxidative addition and transmetallation were detected, and coordination of base to palladium was observed for secondary amines but not triethylamine. Reductive elimination was achieved in the gas phase for a series of para-substituted aryl iodides with phenylacetylene, and the slope of the resulting Hammett plot (ρ) was -0.5. No evidence for the previously hypothesized anionic mechanism was observed. Simultaneous kinetic analysis of charged substrate, products and intermediates in the copper-free Sonogashira reaction was conducted using PSI-ESI-MS and high quality, information rich data for each species over time was obtained. In the absence of protons, reductive elimination is rate-limiting and the rate of reaction is relatively high. In the presence of protons (a byproduct of the reaction), transmetallation is rate-limiting and the rate of reaction is much slower. The use of a strong base was shown to improve the efficiency of the reaction, and an experimentally-derived catalytic cycle for the copper-free Sonogashira reaction is proposed. / Graduate

For the study of homogeneous catalytic reaction mechanisms, the ideal technique would be capable of identifying and measuring in real time the abundances of all components of the reaction mixture, including reactants, products, byproducts, intermediates, and catalyst resting states. This thesis details the development of methodologies designed to transform electrospray ionization mass spectrometry into just such a tool. Species of interests must be charged otherwise invisible in ESI-MS. Therefore, charge-tagged aryl iodide ([4-I-C6H4CH2PPh3]+[Br]-) and a terminal alkyne ([para-(HCC)C6H4CH2PPh3]+[Br]-) were synthesized as the ESI-active substrates for the homogeneous catalysis study. A method named PSI (pressurized sample infusion) was developed to introduce the air and moisture sensitive reaction mixtures to the ESI-MS. The analytical aspects of the method were investigated and optimized. Applicability of the technique was demonstrated through several organic and organometallic mechanism investigations. The above developments were employed to the detailed study of the copper-free Sonogashira (Heck alkynylation) reaction and the hydrodehalogenation of the charged-tag aryl iodide. Simultaneous monitoring of the charged substrate, products and intermediates in the copper-free Sonogashira reaction by PSI-ESI-MS provided rich information about the kinetic and mechanism of this reaction. Kinetic isotope effect study shows a remarkable inverse kinetic isotope effect which is completely unexpected. Numerical models were constructed to simulate the mechanistic observation and to extract the rate constant of each step in the proposed mechanism cycle. The same methodology (PSI technique) was used to the study of the hydrodehalogenation reaction. Key intermediates were detected under the typical reaction conditions. Kinetic isotope effect study was performed in CH3OD and CD3OD. A primary KIE was observed in both deuterated solvents. A revised mechanism cycle was suggested for this reaction based on KIE results, numerical modelling and other experiments. In the proposed cycle deprotonation of methanol occurs on the palladium metal centre instead of the conventional in solution deprotonation (off metal deprotonation). The mechanism of the ligand substitution of charged-tag of a palladium aryl iodide [Pd(TMEDA)(Ar)(I)]+ (Ar = [C6H4CH2PPh3]+[PF6]-) complex against PPh3 was studied in methanol by PSI-ESI-MS. Results revealed that the pathway proceeds quite differently to what had been assumed by others; there was a very fast displacement of [I]– by PPh3 to form [Pd(TMEDA)(Ar)(PPh3)]2+ , followed by a much slower displacement of TMEDA and recoordination of [I]– to form the product [Pd(PPh3)2ArI]+. We successfully integrated UV/Vis spectroscopy, as a complementary method with ESI-MS to shed light into the systems where ESI-MS only is unable to provide a full assignment to homogenous catalysis. The combination of the two fast and sensitive techniques provides a unique opportunity to study the composition of the organometallic reaction mixtures over time. / Graduate / 0486 / zohrabahmadi@gmail.com

The mechanism of a conjugate addition of an alcohol to an alkynic acid ester using a phosphine catalyst was investigated using pressurized sample infusion electrospray ionization mass spectrometry (PSI-ESI-MS) and proton and phosphorus nuclear magnetic resonance (NMR) experiments. Since ESI-MS only detects charged species, and only the phosphonium intermediates and by-products were visible by ESI-MS, 1H NMR was used to track the disappearance of the starting alkyne and the appearance of the conjugate addition product over time. 31P NMR was used to quantify the ESI-MS results. By-product formation was shown to out-compete product formation upon fast addition of alkyne, but with dropwise addition of alkyne, product was shown to dominate. A detailed numerical model was developed using PowerSim software to test mechanistic hypotheses. The experimental results were shown to be consistent with the mechanism proposed by Inanaga, and the cycle was elaborated to account for by-product formation. Piers’catalyst, a ruthenium complex with a phosphonium-functionalized carbene ligand, is a fast-initiating living catalyst for a number of olefin metathesis reactions, including ring-opening metathesis polymerization (ROMP) and cross metathesis (CM). Catalyst speciation was monitored in real-time for the ROMP of norbornene and the CM of 1-hexene using PSI-ESI-MS. The expected mass distribution of charged polymer-catalyst species were not observed, but merely catalyst and decomposition species were visible by ESI-MS. NMR and gel permeation chromatography (GPC) were used to determine quantitatively the presence of polymer and the polydispersity index, respectively. The results suggest that while Piers’ catalyst is indeed fast-initiating, the propagation rate greatly outstrips the initiation rate. In a foray into the area of chemical education, a well-known pH-induced colour change exhibited by the anthocyanins in red cabbage was developed into a simple – and ingestible – classroom demonstration. / Graduate / 0485

This thesis describes research efforts dedicated to the development of palladium(II)-catalyzed oxidative Heck and Heck/Suzuki domino reactions, and the applications of a new microwave heating technology, purpose-built for continuous flow in organic synthesis. Paper I describes the development of a ligand-modulated approach for attaching aryl groups to a chelating vinyl ether. By switching the ligand being used, selectivity for the arylation could be shifted to obtain three different outcomes: internal α- or terminal β-arylation, as well as a serendipitously discovered domino α,β-diarylation process. The latter was proposed to be an effect of para-benzoquinone, effectively acting as a stabilizing π-acidic ligand with the ability to suppress β-hydride elimination. Paper II explores the performance of a new microwave heating technology in combination with continuous flow. The novel nonresonant microwave applicator allowed rapid heating of common laboratory solvents and reaction mixtures above their boiling points with stable and reproducible temperature profiles. The technology was successfully applied to small-scale method development and subsequent scale-out of palladium-catalyzed reactions, heterocycle synthesis and classical organic transformations such as the Fischer indole synthesis. Paper III focuses on developing regioselective oxidative decarboxylative Heck reactions with electron-rich olefins. Successful internal α-arylations were achieved using various olefins and ortho-substituted aromatic acids. The mechanism was also studied by ESI-MS analysis. Key cationic organopalladium intermediates were identified, as well as an unexpected palladium(II)-complex which was isolated and characterized. Its experimentally deduced structure was in accordance with the lowest energy minimum found by DFT calculations. Preliminary findings suggested that the complex acts as a catalyst trap. Paper IV studies the mechanism of the reaction in Paper III by means of DFT calculations. Reductive elimination was identified as the rate-determining step when using a linear enamide as the olefin, due to its propensity to form low energy chelates. Its chelating properties also played a key role in the stability of the isolated palladium(II)-complex. The complex, which can act as a catalyst trap, was characterized by X-ray crystallography.

Dynamic combinatorial chemistry (DCC) is a powerful tool to identify new ligands for biological targets. In the technique, library synthesis and hit identification are neatly combined into a single step. A labile functionality between fragments allows the biological target to self-select binders from a dynamic combinatorial library (DCL) of interconverting building blocks. The scope of suitable reversible reactions that proceed under thermodynamic control in physiological conditions has been gradually expanded over the last decades, however DCC has thus far failed to gain traction as a technique appropriate for drug discovery in the pharmaceutical industry. The constraints placed on library size by validated analytical techniques, and the effort-intensive reality of this academically elegant concept have not allowed DCC to develop into a broad-platform technique to compete with the high-throughput screening campaigns favoured by medicinal chemists. This thesis seeks to develop DCL analysis techniques, in an effort to increase the library size and accelerate the analysis of DCC experiments. Using a 19F-labelled core scaffold, we constructed a DCL that could be monitored non-invasively by 19F NMR. Building on NMR techniques developed by fragment screening and non-biological DCC campaigns, the method was developed to circumvent the undesired equilibrium-perturbing side effects arising from sample-consuming analytical methods. The N-acylhydrazone (NAH) DCL equilibrated rapidly at pH 6.2 using 4-amino-L-phenylalanine (4-APA) as a novel, physiologically benign, nucleophilic catalyst. The DCL was designed to target b-ketoacyl-ACP synthase III (FabH), an essential bacterial enzyme and antibiotic target. From the 5-membered DCL, a single combination was identified as a privileged structure by our 19F NMR method. The result correlated well with an in vitro assay, validating 19F NMR as a tool for DCL screening. During the 19F NMR study we identified an established antimicrobial compound, 4,5- dichloro-1,2-dithiole-3-one (HR45), to have potential as a core scaffold from which to develop future DCLs targeting FabH. Despite the potentially tractable chemistry of HR45 for DCC, lack of knowledge around the inhibitory mechanism of the compound prevented us from proceeding. Thus, we used mass spectrometry, NMR and molecular modelling to show that HR45 acts by forming a covalent adduct with S. aureus FabH. The 5-chloro substituent directs attack from the nucleophilic thiol side chain of the essential active site cysteine-112 residue via a Michael-type addition elimination mechanism. Although interesting, this mechanism disfavoured the use of HR45 as a core scaffold for NAH exchange in a DCC campaign. Electrospray ionisation mass spectrometry (ESI-MS) is a powerful technique that allows for larger DCLs by eliminating the size-limitations imposed by the need for spectral or chromatographic resolution of DCL members. We developed a 4-APAcatalysed NAH library targeting the pyridoxal 5’-phosphate (PLP) dependent enzyme 7,8-diaminopelargonic acid synthase (BioA), an essential enzyme in the biotin biosynthesis pathway. We exploited the aldehyde moiety of PLP to form an NAH DCL with a panel of hydrazides, and used the BioA isozymes from M. tuberculosis (Mtb) and E. coli to template the library. A combination of buffer exchange and denaturing ESI-MS allowed us to conduct a DCC experiment with a 29-member DCL. Hits from the DCC experiment correlated well with differential scanning fluorimetry (DSF) results. Of these hits, 5 compounds were selected for further study. In vivo activity was displayed by 2 compounds against E. coli and the ESKAPE pathogen A. baumannii. The identification of compounds with antibacterial activity from a DCL further validates ESI-MS as a platform technology for drug discovery.

SOUZA, Pedro Filho Noronha de. Análise comparativa de parâmetros bioquímicos e fisiológicos de um genótipo de feijão-de-corda (Vigna unguiculata L. Walp.) suscetível e seu mutante derivado, resistente, infectados com o vírus do mosaico severo do caupi (CPSMV). 2016. 180 f. Tese (Doutorado em bioquímica)- Universidade Federal do Ceará, Fortaleza-CE, 2016. / Submitted by Elineudson Ribeiro (elineudsonr@gmail.com) on 2016-07-28T15:30:39Z No. of bitstreams: 1 2016_tese_pfnsouza.pdf: 6363124 bytes, checksum: a74bcc0c69df39e0286b864e93babb1a (MD5) / Approved for entry into archive by José Jairo Viana de Sousa (jairo@ufc.br) on 2016-08-02T14:43:45Z (GMT) No. of bitstreams: 1 2016_tese_pfnsouza.pdf: 6363124 bytes, checksum: a74bcc0c69df39e0286b864e93babb1a (MD5) / Made available in DSpace on 2016-08-02T14:43:45Z (GMT). No. of bitstreams: 1 2016_tese_pfnsouza.pdf: 6363124 bytes, checksum: a74bcc0c69df39e0286b864e93babb1a (MD5) Previous issue date: 2016 / Cowpea is an important crop that makes major nutritional contributions as a source of proteins and carbohydrates in the diet of many people worldwide. However, its production is impaired due to various stresses including those of biotic origins. Cowpea Severe Mosaic Virus (CPSMV) infects cowpeas leading to severe symptoms and low productivity. Several studies of plant-virus interaction show that seed treatment with Ethyl methanesulfonate (EMS, chemical mutagen), results in a resistant phenotype in plants, which was previously susceptibility, to virus infection of the Potyvirus genus. The aim of this study was to investigate some physiological and biochemical parameters of a susceptible cowpea cultivar (CPI) (CE-31, sin. Pitiuba) in comparison with its derived resistant mutagenized (MCPI), both infected with CPSMV. MCPI plantlets were obtained after treatment of CE-31 seeds with 0.04% EMS. Two different approaches were used in this study: 1) biochemical (antioxidant enzymes and H2O2 content, PR-proteins and secondary metabolites) and physiological analysis (photosynthetic parameters and chlorophyll content); and 2) Label free quantitative proteomic approach (LC-ESI-MS / MS) to identify proteins responsive to viral infection. Our results showed that MCPI had no symptoms of CPSMV infection and biochemical (high H2O2, PR-proteins and secondary compounds [phenolic and lignin]) and physiological responses (High photosynthesis index and chlorophyll content) is activated in MCPI plantlets after CPSMV inoculation. With regard to proteomic analysis, 99 proteins were differentially represented, where these 68 are up- and 31 down represented in MCPI compared to CPI. Regardless whether to CPI (susceptible) or MCPI (mutagenized resistant) plantlets, CPSMV induce changes in proteome profile that involve several biological process (energy and metabolism, photosynthesis, response to stress, oxidative burst, and scavenging). Moreover, these results suggest that the CPSMV responsive proteins in the MCPI represent a complex network involving in resistant mechanisms to CPSMV. Treatment of the susceptible CE-31 genotype seeds with the mutagenic agent EMS induced genomic alterations generating a cowpea mutagenized resistant to CPSMV by apparently inducing classical biochemical and physiological responses against infection. / O feijão-de-corda tem grande importância socioeconômica no Nordeste brasileiro. Entretanto, sua produção é baixa devido a diversos fatores bióticos, como, por exemplo, o vírus do mosaico severo do caupí (CPSMV, gênero Comovirus), que apresenta grande destaque, por causar a virose que mais acomete essa cultura no país. No estudo da interação planta-vírus, diversos trabalhos mostram que o tratamento de sementes com o etil metanosulfonato (EMS, mutagênico químico) resulta no fenótipo de resistência em plantas que, anteriormente, apresentavam susceptibilidade à infecção por vírus do gênero Potyvirus. Por essa razão, o presente estudo teve como objetivo investigar as respostas de defesa bioquímicas e fisiológicas das plantas de feijão-de-corda do genótipo (CE-31) susceptível ao CPSMV (CPI) a partir de sementes tratadas com EMS (0,04% v/v), e avaliar se as plantas mutagenizadas (MCPI), produzidas a partir dessas sementes se tornaram resistentes ao CPSMV. Duas diferentes abordagens foram utilizadas neste trabalho: 1) análises bioquímicas (enzimas antioxidantes e conteúdo de H2O2, PR-proteínas e compostos secundários) e fisiológicas (parâmetros fotossintéticos e teor de clorofila); 2) abordagem proteômica quantitativa (LC-ESI-MS/MS), livre de marcação, para identificar proteínas responsivas à infecção viral. Os resultados obtidos demonstram que as plantas MCPI são capazes de induzir respostas bioquímicas (aumento de H2O2, indução de PR-proteínas e aumento no conteúdo de compostos secundários) e alterações nos parâmetros fisiológicos (alta taxa fotossintética e teor de clorofila) que, aparentemente, têm relação com o fenótipo de resistência das plantas mutagenizadas ao CPSMV. Na análise proteômica, 99 proteínas foram identificadas como sendo diferenciais, das quais 68 aumentaram e 31 diminuíram em abundância nas plantas MCPI em relação as plantas CPI. A análise proteômica, mostrou diversas vias metabólicas (Metabolismo Redox, Energia e Metabolismo, Fotossíntese, Metabolismo de RNA e Defesa) envolvidas nas respostas de defesa das plantas MCPI frente a infecção viral. O tratamento das sementes com o EMS, resultou em plantas de feijão-de-corda com fenótipo de resistência capazes de acionar mecanismos de defesa para impedir a infeção viral.

Submitted by VIVIANE MAGRINI null (viviane_magrini@yahoo.com.br) on 2016-03-01T20:13:04Z No. of bitstreams: 1 Viviane Magrini.pdf: 5645437 bytes, checksum: 25fafce9b5405010cb429b17c172011c (MD5) / Approved for entry into archive by Juliano Benedito Ferreira (julianoferreira@reitoria.unesp.br) on 2016-03-03T19:58:52Z (GMT) No. of bitstreams: 1 magrini_v_me_araiq_par.pdf: 1328742 bytes, checksum: 4a2b2b27d644fb4d71ce706407afdc38 (MD5) / Made available in DSpace on 2016-03-03T19:58:52Z (GMT). No. of bitstreams: 1 magrini_v_me_araiq_par.pdf: 1328742 bytes, checksum: 4a2b2b27d644fb4d71ce706407afdc38 (MD5) Previous issue date: 2016-02-18 / Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) / O gênero Calycophyllum pertence à família Rubiaceae, conhecida pela diversidade de metabólitos secundários, e pela variedade de atividades biológicas: antifúngica, bactericida, antiviral e inseticida. A espécie vegetal Calycophyllum spruceanum Benth (Rubiaceae) é muito utilizada pelas populações da América do Sul no combate a doenças dermatológicas, estomacais, diabetes, parasitoses, câncer, entre outras, despertando o interesse pela investigação química deste táxon. A presente pesquisa objetivou o estudo bio-guiado dos extratos obtidos das folhas, galhos e de cascas do caule de C. spruceanum visando à identificação das substâncias bioativas isoladas, incluindo as inativas. A meta é identificar nas substâncias isoladas aquelas com propriedades cosméticas associadas ao uso popular desta espécie, conhecida na região Amazônica por esta propriedade. Diferentes extratos foram preparados e analisados por Cromatografia Líquida de Alta Eficiência (CLAE) e Espectroscopia de Ressonância Magnética Nuclear (RMN 1H), acompanhados por ensaios biológicos (ensaio de capacidade redutora dos radicais DPPH˙ e ABTS˙+) para determinação de atividade antioxidante. O direcionamento do estudo foi feito em função dos ensaios antioxidantes aplicados aos extratos, conduzindo o estudo experimental para o extrato hidroetanólico de folhas ao foco do estudo, por ter apresentado a maior atividade antioxidante. Por meio de métodos espectroscópicos e espectrométricos (CLAE-DAD, CLAE-EM, CLAE-EM/EM; RMN de 1H e 13C; TOCSY 1D; HMBC; EM-IES) deste extrato e respectivas frações, pode-se identificar algumas substâncias antioxidantes potencialmente ativas, sendo estas: ácido 3,5-di-O-E-cafeoilquínico; duas séries homólogas de proantocianidinas (tipo-B) com grau de polimerização de 1 a 4, e proantocianidinas monoglicosiladas (tipo-B O-glicosilada) com grau de polimerização de 1 a 3; ácido 1,4-di-O-E-cafeoilquínico, quercetina 3-O-α-arabinopiranosil (16) β-glucopiranosídeo, ácido cafeoilquínico e canferol 3-O-β-D-glicopiranosil (12) β-D-xilopiranosídeo. Espera-se que ao final deste estudo, as informações forneçam subsídios para uma possível aplicação dos resultados científicos sobre a espécie vegetal, visando um produto para a linha de cosmético funcional. / The Calycophyllum genus belongs to the Rubiaceae’s family, known for its high diversity of secondary metabolites, which have a wide range of biological activities (antifungal, antibacterial, antiviral and pesticide). It is widely used by the people of South America against skin diseases, stomach, diabetes, parasites, cancer, among others. It is therefore essential to study the chemical composition of these species used in folk medicine, aiming to validate the traditional use, with scientific data and thus contributing to the search for new bioproducts. This research aimed the bio-guided fractionation of the extracts from the leaves, branches and stem bark extracts of Calycophyllum spruceanum Benth (Rubiaceae), to identify the secondary metabolites in the extracts and fractions, including bioactive ones, for further evaluation of the cosmetic potential of this species, commonly known in the Amazon region. Different extracts were prepared and analyzed by High Performance Liquid Chromatography (HPLC) and Nuclear Magnetic Resonance spectroscopy (1H NMR), and screened by biological assays (assay of reducing capability of ABTS˙+ and DPPH˙radicals) to determine the antioxidant activity. The chemical investigation was carried out based on the antioxidant activity of the extracts. The experimental study focused on the hydroethanolic extract of leaves, that presented the higher antioxidant activity, which would support the traditional uses of this specie as cosmetic. By means spectroscopic and spectrometric analysis (HPLC-DAD, HPLC-MS, HPLC-MS/MS; 1H NMR and 13C; TOCSY 1D; HMBC, ESI-MS) of this extract and its fractions, some compounds were identified: the 3,5-di-O-E-caffeoylquinic acid; two homologous series of proanthocyanidins (B-type) with a degree of polymerization from 1 to 4 and monoglicosyl proanthocyanidins (B-type O-glycosylated) with degree of polymerization from 1 to 3; 1,4-di-O-E-caffeoylquinic acid, quercetin-3-O-α-arabinopyranosyl (16) β-glucopyranoside, caffeoylquinic acid and kaempferol 3-O-β-D-glucopyranosyl (12) β-D-xilopyranosil, which could be responsible for the activity. It is expected that the the complete study of this species provide chemical information, which will validate this plant specie as a potential source of antioxidant natural products that could be material for functional cosmetics. / CNPq: 166069/2014-0

Made available in DSpace on 2014-06-12T23:16:16Z (GMT). No. of bitstreams: 2 arquivo958_1.pdf: 2790418 bytes, checksum: 41ba3566a5e13d0a2496b830fee53158 (MD5) license.txt: 1748 bytes, checksum: 8a4605be74aa9ea9d79846c1fba20a33 (MD5) Previous issue date: 2011 / Conselho Nacional de Desenvolvimento Científico e Tecnológico / Os antibióticos são substâncias químicas que combatem ou inibem o crescimento de organismos, sendo muito utilizados para o controle de certas doenças de origem bacteriana. Faz-se necessário atestar a qualidade destes produtos, uma vez que se torna cada vez mais crescente o seu uso. Além disso, resíduos de antibióticos podem permanecer em alimentos de origem animal, acima de valores considerados seguros quando não são respeitadas as boas práticas veterinárias. Neste trabalho foram desenvolvidos e validados métodos para a determinação de antibióticos em formulações farmacêuticas e em leite. No que diz respeito às formulações farmacêuticas foi desenvolvido um procedimento de análises em fluxo com multicomutação baseado no conceito de fluxo-batelada para a determinação espectrofotométrica de amoxicilina. O procedimento foi baseado na reação da orto-nitroanilina diazotizada em meio alcalino resultando em um sal de diazônio e posterior acoplamento com a amoxicilina para a reação de azo acoplamento. O módulo de análises foi constituído por duas minibombas solenóide, uma válvula de estrangulamento e três válvulas solenóide de três vias, sendo os dispositivos ativos controlados por um microcomputador equipado com uma interface PCL-711S, empregando um programa escrito em linguagem QuickBASIC 4.5. As soluções de amostras e de reagentes foram introduzidas em uma câmara de reação onde foi fixado um LED (λ=435 nm) usado como fonte luminosa e um fototransistor (Til78) usado como detector. As soluções de referência foram estudadas na faixa de 25 a 400 mg L-1 de amoxicilina, com limite de detecção de 5,1 mg L-1, desvio padrão relativo de 3,9% (n=10) e frequência de amostragem de 50 determinações por hora. Foi também desenvolvido um método para determinação de amoxicilina, ampicilina, tetraciclina, oxitetraciclina e cloranfenicol em amostras de leite, empregando o procedimento QuEChERS para o preparo da amostra e de cromatografia líquida acoplada ao espectrômetro de massas no modo de ionização electrospray (LC-ESI-MS) para a identificação e quantificação. Foi utilizada uma coluna C18 e a fase móvel foi composta de água e metanol, com eluição por gradiente. O método foi validado e aplicado para análises de amostras de leites integral e in natura provenientes da bacia leiteira do estado de Pernambuco. A linearidade da curva analítica foi de 20,0 a 400,0 μg L-1 para a tetraciclina e oxitetraciclina. A partir de 2,0 μg L-1 até 12,0 μg L-1 para amoxicilina e ampicilina e entre 0,3 e 1,3 μg L-1 para o cloranfenicol. Os resultados do teste de recuperação variaram na faixa de 83 a 92 %. Pôde-se observar que das dez diferentes marcas de leite pasteurizado integral (tipo longa vida) analisados, em duas foram registradas a presença de amoxicilina e oxitetraciclina. Para as vinte e cinco amostras de leite in natura, pôde-se verificar a presença de amoxicilina e ampicilina em três amostras de leite e a presença de oxitetraciclina e tetraciclina em mais duas amostras. Os valores de resíduos de antibióticos encontrados, em todas as amostras, estavam abaixo do LMR permitido pela legislação vigente

Ring closing metathesis (RCM) is a chemical transformation that converts a bisalkene compound into a cycloalkene. It is catalyzed by transition metal complexes containing carbene ligands (that feature metal-carbon double bonds). The mechanism is well-understood, however, there are numerous details of the reaction that are less well understood, especially concerning catalyst activation and decomposition and formation of byproducts. This thesis takes a new approach to the study of RCM: analysis of the reaction using real-time mass spectrometric techniques. Electrospray ionization (ESI) mass spectrometry was employed in this study, and the real-time aspect was enabled by using pressurized sample infusion (PSI). Observation of the reactants and products was enabled using charge-tagged bis-alkenes of the general formula [Bu2N{(CH2)nCH=CH2}2]+ [PF6]–. These were synthesized in two steps using a generally applicable methodology to generate a wide range of ring sizes of the product, from 5- to 15-membered rings. Examination of their behavior under carefully optimized RCM conditions using Grubbs’ second-generation catalyst showed a wide variation in reaction rates and amount of byproducts, largely due to ring-strain effects (especially high for 5- and 9-membered rings). Byproducts always exhibited a 14 Da mass unit difference from starting materials or products, and Orbitrap MS analysis confirmed it was CH2. Isomerization was suspected to lead to byproducts. A pathway for byproducts via isomerization and cross metathesis was proposed. The source of actual isomerization catalyst was believed to be from the precatalyst itself as the evidence of precatalyst decomposition was observed. Finally, to prove our isomerization hypothesis, an authentic isomerization catalyst was deliberately added into a fast and clean reaction along with Grubbs’ second-generation catalyst, and it produced the expected byproducts. Only small amounts of oligomeric intermediates were observed, probably because of the low concentrations used. [ClPCy3]+ was a new short-lived decomposition product stemming from catalyst breakdown, along with already-known imidazolium and protonated phosphine decomposition products. Overall, the thesis provides deep new insights into the nature of RCM reactions, in particular revealing the importance of isomerization in RCM reactions that are slow due to ring strain effects and in uncovering a new decomposition pathway for important RCM catalysts. / Graduate

Ces travaux de thèse concernent les développements d'une méthode de séparation chromatographique couplée simultanément à l'ESI-MS et l'ICP-MS afin de réaliser l'analyse de spéciation exhaustive des lanthanides en phase aqueuse représentative des phases de désextraction des procédés de traitement du combustible usé. Cette méthode analytique permet de séparer, caractériser et quantifier des complexes de lanthanides à ligands polyaminocarboxyliques comme le DTPA et l'EDTA, utilisés comme agents complexants dans ces procédés. La méthode de séparation par chromatographie HILIC des complexes de lanthanides a été mise au point avec la phase stationnaire à fonctions amide. Un criblage d'une large gamme de compositions de phase mobile a permis de déterminer que le mécanisme d'adsorption est prédominant lors l'élution des complexes de lanthanides et d'obtenir des conditions de séparation optimisées. Des conditions d'analyse plus rapides obtenues avec une colonne à fonctions amide de granulométrie sub-2 µm et de longueur plus faible ont permis de réduire le temps d'analyse d'un facteur 2,5 et la consommation de solvant de 25 %. La caractérisation structurale et isotopique par HILIC ESI-MS a été réalisée ainsi que la mise au point d'une méthode d'étalonnage externe. Les performances analytiques de la méthode de quantification ont été déterminées. Enfin, le développement d'un système de couplage de l'HILIC à l'ESI-MS et l'ICP-MS a été réalisé. Une méthode de quantification simultanée par ESI-MS et par ICP-MS a permis de déterminer la distribution quantitative des espèces en solution ainsi que les performances analytiques associées. / This work focuses on the development of a chromatographic separation method coupled to both ESI-MS and ICP-MS in order to achieve the comprehensive speciation analysis of lanthanides in aqueous phase representative of back-extraction phases of advanced spent nuclear fuel treatment processes. This analytical method allowed the separation, the characterization and the quantitation of lanthanides complexes holding polyaminocarboxylic ligands, such as DTPA and ETDA, used as complexing agents in these processes. A HILIC separation method of lanthanides complexes has been developed with an amide bonded stationary phase. A screening of a wide range of mobile phase compositions demonstrated that the adsorption mechanism was predominant. This screening allowed also obtaining optimized separation conditions. Faster analysis conditions with shorter amide column packed with sub 2 µm particles reduced analysis time by 2.5 and 25% solvent consumption. Isotopic and structural characterization by HILIC ESI-MS was performed as well as the development of external calibration quantitation method. Analytical performances of quantitation method were determined. Finally, the development of the HILIC coupling to ESI-MS and ICP-MS was achieved. A simultaneous quantitation method by ESI-MS and ICP-MS was performed to determine the species quantitative distribution in solution. Analytical performances of quantitation method were also determined.