A mass spectrometry study of compounds with environmental and biological interest

Tese de doutoramento, Química (Química Analítica), Universidade de Lisboa, Faculdade de Ciências, 2010The leading thread of this work is the application of Mass Spectrometry to the study of environmental and biological relevant compounds. Several studies were performed involving the development and optimization of efficient analysis methodologies, the behaviour of the compounds under study was investigated using various mass spectrometry techniques and fundamental studies were carried out, most of them complemented with semi-empirical calculations. Matrix-Assisted Laser Desorption/Ionization (MALDI) has been extensively used for the analysis of large molecules. Nevertheless, since the technique provides soft and efficient ionization of thermolabile and non-volatile organic compounds, we decided to investigate its application to small molecules as an alternative ionization method. For that we compared the performance of nanosized TiO2 anatase as matrix with that of common organic matrices. The formation of organic matrix-flavonoid cluster ions was also investigated and it was found to be dependent of the structure of the flavonoid. Aniline derivatives are of great industrial importance and several constitute a significant group of pollutants. The gas-phase behaviour of halo- and nitroanilines under electrospray ionization mass spectrometry conditions was studied. Isomer identification methodologies are proposed using for that purpose competitive fragmentations of protonbound heterodimers. Preliminary results on the behaviour of the aniline derivatives when subjected to electron capture dissociation conditions are presented and discussed. The behaviour of five isoflavones was studied under electrospray ionization mass spectrometry in the positive ion mode. The fragmentation mechanisms were proposed taking into account MSn experiments, accurate mass measurements and semi-empirical calculations. Some of the fragmentations were found to be dependent on the substitution pattern of the B-ring and the ions afforded by these fragmentations can be considered as diagnostic ions. Several newly synthesized -lactones, fused or C-C linked to sugar rings, were studied. Their fragmentation mechanisms were proposed taking into account MSn experiments and semi-empirical calculations. FTICR-MS; ESI; MALDI; Fragmentation Mechanisms; Collision Induced DissociationEste trabalho tem como domínio comum a aplicação da Espectrometria de Massa ao estudo de vários compostos com interesse ambiental e biológico. Foram realizados estudos que envolvem o desenvolvimento e optimização de metodologias eficientes de análise; foi investigado o comportamento dos compostos em estudo usando várias técnicas de espectrometria de massa e foram realizados estudos de natureza fundamental, a maioria dos quais complementados por cálculos semi-empíricos. Concretamente: Ionização/desorção laser assistida por matriz (MALDI) é uma técnica de ionização muito usada na análise de moléculas com massas moleculares elevadas. No entanto, a sua capacidade de ionizar de um modo suave e eficiente compostos não voláteis e termicamente instáveis levou-nos a investigar a sua aplicabilidade à análise de moléculas pequenas. Para tal, comparou-se a performance da fase anatase do TiO2 com a de matrizes orgânicas convencionais na ionização de vários compostos cobrindo várias classes. Um dos compostos em particular, um flavonóide, formou iões agregados com a matriz orgânica, o que nos levou a aprofundar o estudo deste comportamento. Conclui-se que a estrutura do flavonóide influencia a formação dos iões agregados. Derivados de anilina têm uma grande importância a nível industrial e os derivados halogenados, em particular, constituem um grupo de poluentes bastante significativo. Assim, estudou-se o comportamento em fase gasosa de halo- e nitroanilinas com o intuito de estabelecer mecanismos de fragmentação e metodologias que permitam a identificação de isómeros. Para a identificação de isómeros recorreu-se a fragmentações competitivas de heterodímeros ligados por protão. Também são apresentados e discutidos resultados preliminares sobre o comportamento de derivados de anilina quando expostos a condições de dissociação por captura de electrões. O comportamento de cinco isoflavonas foi estudado no modo positivo e recorrendo à espectrometria de massa com ionização por electrospray. Foram propostos mecanismos de fragmentação tendo em conta experiências de espectrometria de massa tandem, medições de massa exacta e cálculos semi-empíricos. Algumas das fragmentações observadas são dependentes do anel B e dos seus substituintes e, como tal, os iões originados por estas fragmentações podem ser considerados como iões diagnóstico. Várias -lactonas fundidas ou ligadas a açúcares, compostos recentemente sintetizados, foram estudadas com o intuito de estabelecer mecanismos de fragmentação. Tal foi feito tendo em conta dados de espectrometria de massa tandem e cálculos semi-empíricos. FTICR-MS; ESI; MALDI; Mecanismos de Fragmentação; Dissociação Induzida por ColisãoThe work presented in this thesis was performed in the Environmental and Biological Mass Spectrometry Group, Departamento de Química e Bioquímica da Faculdade de Ciências da Universidade de Lisboa, with the financial support of Fundação para a Ciência e a Tecnologia (SFRH/BD/27614/2006

Design and anticancer properties of new water-soluble ruthenium–cyclopentadienyl complexes

Ruthenium complexes are emerging as one of the most promising classes of complexes for cancer therapy. However, their limited aqueous solubility may be the major limitation to their potential clinical application. In view and to contribute to the progress of this field, eight new water-soluble Ru(II) organometallic complexes of general formula [RuCp(mTPPMS)n(L)] [CF3SO3], where mTPPMS = diphenylphosphane-benzene-3-sulfonate, for n = 2, L is an imidazole-based ligand (imidazole, 1-benzylimidazole, 1-butylimidazole, (1-(3 aminopropyl)imidazole), and (1-(4-methoxyphenyl)imidazole)), and for n = 1, L is a bidentate heteroaromatic ligand (2-benzoylpyridine, (di(2-pyridyl)ketone), and (1,2-(2-pyridyl)benzo-[b]thiophene)) were synthesized and characterized. The new complexes were fully characterized by NMR, FT-IR, UV–vis., ESI-HRMS, and cyclic voltammetry, which confirmed all the proposed molecular structures. The antiproliferative potential of the new Ru(II) complexes was evaluated on MDAMB231 breast adenocarcinoma, A2780 ovarian carcinoma, and HT29 colorectal adenocarcinoma cell lines, showing micromolar (MDAMB231 and HT29) and submicromolar (A2780) IC50 values. The interaction of complex 6 with human serum albumin (HSA) and fatty-acid-free human serum albumin (HSAfaf) was evaluated by fluorescence spectroscopy techniques, and the results revealed that the ruthenium complex strongly quenches the intrinsic fluorescence of albumin in both cases.info:eu-repo/semantics/publishedVersio

Biological activity and cellular uptake of [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] complex

Anticancer activity of the new [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] (Me(2)bpy = 4,4'-dimethyl-2,2'-bipyridine) complex was evaluated in vitro against several human cancer cell lines, namely A2780, A2780CisR, HT29, MCF7, MDAMB231 and PC3. Remarkably, the IC50 values, placed in the nanomolar and sub-micromolar range, largely exceeded the activity of cisplatin. Binding to human serum albumin, either HSA (human serum albumin) or HSA(faf) (fatty acid-free human serum albumin) does not affect the complex activity. Fluorescence studies revealed that the present ruthenium complex strongly quench the intrinsic fluorescence of albumin. Cell death by the [Ru(eta(5)-C5H5)(PPh3)(Me(2)bpy)][CF3SO3] complex was reduced in the presence of endocytosis modulators and at low temperature, suggesting an energy-dependent mechanism consistent with endocytosis. On the whole, the biological activity evaluated herein suggests that the complex could be a promising anticancer agent. (C) 2013 Elsevier Inc. All rights reserved

Synthesis of organometallic Ruthenium(II) complexes with strong activity against several human canceer cell lines

A new family of "RuCp" (Cp=eta(5)-C5H5) derivatives with bidentate N,O and N,N'-heteroaromatic ligands revealed outstanding cytotoxic properties against several human cell lines namely, A2780, A2780CisR, HT29, MCF7, MDAMB231, and PD. IC50 values were much lower than those found for cisplatin. Crystal structure of compound 4 was determined by X-ray diffraction studies. Density functional theory (DFT) calculations performed for compound 1 showed electronic flow from the ruthenium center to the coordinated bidentate ligand, in agreement with the electrochemical studies and the existence of a metal-to-ligand charge-transfer (MLCT) band evidenced by spectroscopic data

Synthesis and structural characterization of new Piano-stool Ruthenium(II) complexes bearing 1-Butylimidazole heteroaromatic ligand

New cationic ruthenium(II) complexes with the formula [Ru(eta(5)-C5H5)(LL)(1-BuIm)] [Z], with (LL) = 2PPh(3) or DPPE, and Z = CF3SO3-, PF6-, BPh4-, have been synthesized and fully characterized. Spectroscopic and electrochemical studies revealed that the electronic properties of the coordinated 1-butylimidazole were clearly influenced by the nature of the phosphane coligands (LL) and also by the different counter ions. The solid state structures of the six complexes determined by X-ray crystallographic studies, confirmed the expected distorted three-legged piano stool structure. However the geometry of the 1-butylimidazole ligand was found considerably different in all six compounds, being governed by the stereochemistry of the mono and bidentate coligands (PPh3 or DPPE)

New water-soluble Ruthenium(II) cytotoxic complex: biological activity and celular distribution

A novel water soluble organometallic compound, [RuCp(mTPPMSNa)(2,2'-bipy)][CF3SO3] (TM85, where Cp=eta(5)-cyclopentadienyl, mTPPMS = diphenylphosphane-benzene-3-sulfonate and 2,2'-bipy = 2,2'-bipyridine) is presented herein. Studies of interactions with relevant proteins were performed to understand the behavior and mode of action of this complex in the biological environment. Electrochemical and fluorescence studies showed that TM85 strongly binds to albumin. Studies carried out to study the formation of TM85 which adducts with ubiquitin and cytochrome c were performed by electrospray ionization mass spectrometry (ESI-MS). Antitumor activity was evaluated against a variety of human cancer cell lines, namely A2780, A2780cisR, MCF7, MDAMB231, HT29, PC3 and V79 non-tumorigenic cells and compared with the reference drug cisplatin. TM85 cytotoxic effect was reduced in the presence of endocytosis modulators at low temperatures, suggesting an energy-dependent mechanism consistent with endocytosis. Ultrastructural analysis by transmission electron microscopy (TEM) revealed that TM85 targets the endomembranar system disrupting the Golgi and also affects the mitochondria. Disruption of plasma membrane observed by flow cytometry could lead to cellular damage and cell death. On the whole, the biological activity evaluated herein combined with the water solubility property suggests that complex TM85 could be a promising anticancer agent. (C) 2013 Elsevier Inc. All rights reserved

Gas-phase interaction between nickel (II) and nitrobenzyl azides: An ESI-MSn study

Organoazides are intermediates in the synthesis of imidometal complexes. Metalimido complexes have an important role in catalysis. Thus, the organoazide interaction with metals is an important issue. However, the isolation of organoazidometal complexes is difficult due to the easy loss of N-2. The present study describes the complexation of nickel by nitrobenzyl azides by means of electrospray ionization mass spectrometry (ESI-MS), which was used also as a probe for the characterization of isomers. Organoazidometal complexes were observed and isolated from solutions of NiCl2 and NiBr2 in methanol/water. A different solvent, ethanol/water was also used. The complexes detected were singly and doubly positively charged, with various stoichiometries. The most abundant species were [Ni(II)Az(3)](2+) for ortho- and para-isomers, and [Ni(II)Az(3)(H2O)](2+) for meta isomer, where Az stands for nitrobenzyl azides. The ortho isomer showed several single positively charged complexes integrating chloride as a ligand. The mass spectra of the three isomers allowed their differentiation based on different behaviour in the mass range m/z 296 m/z 312. The MS2 spectra of [Ni(II)Az(3)](2+) were investigated aiming to characterize the three isomers but this analysis was not absolutely conclusive about the coordination site(s). Density functional theory calculations provided possible structures for the [Ni(II)Az(3)](2+) cation with the different isomers and their coordination modes could be responsible for their fragmentation pathways. (C) 2013 Elsevier B.V. All rights reserved