M. Paula M. Marques

Analytical Approaches toward Successful Human Cell Metabolome Studies by NMR Spectroscopy

The aim of this work was to investigate the effects of cell handling and storage on cell integrity and (1)H high resolution magic angle spinning (HRMAS) NMR spectra. Three different cell types have been considered (lung tumoral, amniocytes, and MG-63 osteosarcoma cells) in order for sample-dependent effects to be identified. Cell integrity of fresh cells and cells frozen in cryopreservative solution was approximately 70-80%, with the former showing higher membrane degradation, probably enzymatic, as indicated by increased phosphocholine (PC) and/or glycerophosphocholine (GPC). Unprotected freezing (either gradual or snap-freezing) was found to lyse cells completely, similar to mechanical cell lysis. Besides enhanced metabolites visibility, lysed cells showed a different lipid profile compared to intact cells, with increased choline, PC, and GPC and decreased phosphatidylcholine (PTC). Cell lysis has, therefore, a significant effect on cell lipid composition, making handling reproducibility an important issue in lipid analysis. Sample spinning was found to disrupt 5-25% of cells, depending on cell type, and HRMAS was shown to be preferable to solution-state NMR of suspensions or supernatant, giving enhanced information on lipids and comparable resolution for smaller metabolites. Relaxation- and diffusion-edited NMR experiments gave limited information on intact cells, compared to lysed cells. The (1)H HRMAS spectra of the three cell types are compared and discussed.

Experimental and Theoretical Evidence of CH⋅⋅⋅O Hydrogen Bonding in Liquid 4-Fluorobenzaldehyde

The presence of C-H...O hydrogen bonds in liquid 4-fluorobenzaldehyde has been studied by a combination of theoretical and spectroscopic methods. Ab initio calculations yielded bond energies and preferred bonding geometries, and the calculated spectroscopic properties have been compared with the experimental results. The presence of C-H...O hydrogen bonds in the liquid phase is strongly supported by vibrational and NMR spectroscopic data. Particular attention is paid to the spectroscopic effects related to the predicted shortening of the C-H bond engaged in the C-H...O contact. The concentration-dependent intensity in the C-H stretching region is tentatively assigned to a blue-shift effect due to C-H...O hydrogen bonding.

Decavanadate, decaniobate, tungstate and molybdate interactions with sarcoplasmic reticulum Ca2+-ATPase : quercetin prevents cysteine oxidation by vanadate but does not reverse ATPase inhibition

Recently we demonstrated that the decavanadate (V(10)) ion is a stronger Ca(2+)-ATPase inhibitor than other oxometalates, such as the isoelectronic and isostructural decaniobate ion, and the tungstate and molybdate monomer ions, and that it binds to this protein with a 1 : 1 stoichiometry. The V(10) interaction is not affected by any of the protein conformations that occur during the process of calcium translocation (i.e. E1, E1P, E2 and E2P) (Fraqueza et al., J. Inorg. Biochem., 2012). In the present study, we further explore this subject, and we can now show that the decaniobate ion, [Nb(10) = Nb(10)O(28)](6-), is a useful tool in deducing the interaction and the non-competitive Ca(2+)-ATPase inhibition by the decavanadate ion [V(10) = V(10)O(28)](6-). Moreover, decavanadate and vanadate induce protein cysteine oxidation whereas no effects were detected for the decaniobate, tungstate or molybdate ions. The presence of the antioxidant quercetin prevents cysteine oxidation, but not ATPase inhibition, by vanadate or decavanadate. Definitive V(IV) EPR spectra were observed for decavanadate in the presence of sarcoplasmic reticulum Ca(2+)-ATPase, indicating a vanadate reduction at some stage of the protein interaction. Raman spectroscopy clearly shows that the protein conformation changes that are induced by V(10), Nb(10) and vanadate are different from the ones induced by molybdate and tungstate monomer ions. Here, Mo and W cause changes similar to those by phosphate, yielding changes similar to the E1P protein conformation. The putative reduction of vanadium(V) to vanadium(IV) and the non-competitive binding of the V(10) and Nb(10) decametalates may explain the differences in the Raman spectra compared to those seen in the presence of molybdate or tungstate. Putting it all together, we suggest that the ability of V(10) to inhibit the Ca(2+)-ATPase may be at least in part due to the process of vanadate reduction and associated protein cysteine oxidation. These results contribute to the understanding and application of these families of mono- and polyoxometalates as effective modulators of many biological processes, particularly those associated with calcium homeostasis.

Effect of the Metal Center on the Antitumor Activity of the Analogous Dinuclear Spermine Chelates (PdCl2)2(Spermine) and (PtCl2)2(Spermine)

The cytotoxic activity of (PdCl2)2(spermine) against a human cancer cell line (HSC-3) was evaluated. The re- sults show an activity higher than that of the analogous Pt(II) chelate and, for 24 h incubations, identical to that of cis- platin. They also suggest a faster intracellular uptake and DNA binding relative to cisplatin.

Study of Copper(II) Polyazamacrocyclic Complexes by Electronic Absorption Spectrophotometry and EPR Spectroscopy

Copper(II) mononuclear complexes with the polyazamacrocyclic ligands NOTA (1,4,7-triazacyclononane-N,N′,N″triacetate), DOTA (1,4,7,10-tetraazacyclo-dodecane-N,N′,N″,N″′-tetraacetate), NOTP [1,4,7-triazacyclononane-N,N′,N″-tris(methylenephosphonate)] and DOTP [1,4,7,10tetraazacyclododecane-N,N′,N″,N‴-tetrakis(methylenephosphonate)] were characterised in aqueous solution by electronic absorption spectrophotometry and electron paramagnetic resonance (EPR) spectroscopy. While two isomeric species were detected by EPR spectroscopy in frozen samples of the polycarboxylate complexes, only one complex form was found for the poly(methylenephosphonate) systems. The results obtained are in accordance with a six-coordinate pattern for the metal centre, yielding tetragonally distorted complexes in solution, in agreement with previously reported studies on CuII complexes, both in solution and in the solid state.

Guanine: A Combined Study Using Vibrational Spectroscopy and Theoretical Methods

The present paper reports a conformational study of solid-state anhydrous guanine, using vibrational spectroscopy techniques—infrared, Raman, and inelastic neutron scattering—coupled to quantum mechanical methods at the DFT level, both for the isolated molecule and the condensed state. In both cases, the 7H-keto-amino tautomer was found to be the prevalent form, contrary to aqueous solutions and hydrated polycrystalline guanine, where the 9H-keto-amino tautomer is the most favoured species. This paper is a significant contribution for the existing spectroscopic characterization of this purine base, by unambiguously assigning its vibrational spectra.

Polymorphism in Cisplatin Anticancer Drug

This study reports a combined experimental and theoretical study of the solid-state polymorphism of the anticancer agent cisplatin. A complete assignment was performed for the inelastic neutron scattering (INS) and Raman spectra collected simultaneously for cisplatin, at different temperatures, with a view to obtain reliable and definitive evidence of the relative thermal stability of its α and β polymorphic species. A marked temperature-dependent hysteresis was observed, as previously reported in the literature. Theoretical calculations were carried out at the density functional theory level, using a plane-wave basis set approach and pseudopotentials. A detailed comparison with the experimental Raman and INS data showed that the α polymorph is present at the lowest temperatures, whereas the β form occurs near room temperature. Furthermore, regions of coexistence of both forms are identified, which depend on the working mode (heating or cooling). These findings imply that Raman spectroscopy allows clear identification of the α and β polymorphs at a given temperature and can unambiguously discriminate between them. Elucidation of the polymorphic equilibrium of this widely used anticancer drug is paramount for its pharmaceutical preparation and storage conditions.

Inelastic Neutron Scattering Study of PtII Complexes Displaying Anticancer Properties

The well-known platinum(II) chemotherapeutic drugs cisplatin [cis-(NH(3))(2)PtCl(2)] and carboplatin [Pt(NH(3))(2)C(6)O(4)H(6)], as well as the analogous transplatin [trans-(NH(3))(2)PtCl(2)], were studied by inelastic neutron scattering (INS) spectroscopy, coupled to quantum mechanical methods, and some ancillary work with X-ray diffraction on powders. An assignment of the experimental spectra was carried out based on the calculated INS transition frequencies and intensities (at the DFT level), thereby achieving a good correspondence between the calculated and observed data. Unusually good-quality INS spectra were obtained from about 250 mg, which is the smallest sample of a hydrogenous compound for which a successful INS interpretation has been reported. The knowledge of the local configuration of this kind of complexes is essential for an accurate understanding of their activity, which will pave the way for the rational design of novel third-generation drugs comprising cisplatin- and carboplatin-like moieties.

Identification of synthetic precursors of amphetamine-like drugs using Raman spectroscopy and ab initio calculations: β-Methyl-β-nitrostyrene derivatives

The present work reports a vibrational spectroscopic study of several beta-methyl-beta-nitrostyrene derivatives, which are important intermediates in the synthesis of illicit amphetamine-like drugs, such as 3,4-methylenedioxymethamphetamine (MDMA), 3,4-methylenedioxyamphetamine (MDA), p-methoxyamphetamine (PMA) and 4-methylthioamphetamine (4-MTA). A complete conformational analysis of 3,4-methylenedioxy-beta-methyl-beta-nitrostyrene (3,4-MD-MeNS), 4-methoxy-beta-methyl-beta-nitrostyrene (4-MeO-MeNS), 4-methylthio-beta-methyl-beta-nitrostyrene (4-MeS-MeNS), was carried out by Raman spectroscopy coupled to ab initio MO calculations--both complete geometry optimisation and harmonic frequency calculation. The Raman spectra show characteristic features of these precursors, which allow their ready differentiation and identification. It was verified that the conformational behaviour of these systems is mainly determined by the stabilising effect of pi-electron delocalisation.

Fluoxetine and Norfluoxetine Revisited: New Insights into the Electrochemical and Spectroscopic Properties

The extent to which humans and wildlife are exposed to the vast array of anthropogenic chemicals and their degradation products, along with related naturally occurring compounds, is nowadays an important issue. The study of the physical-chemical properties of the compounds and/or degradation products is an important subject because some of them are intrinsically related to its resistance to degradation and/or bioaccumulation. Accordingly, the study of the electrochemical behavior of the selective serotonin reuptake inhibitor fluoxetine and its main metabolite norfluoxetine was investigated. The identification of the oxidation processes was done via two fluoxetine analogues, 1-(benzyloxy)-4-(trifluoromethyl)benzene and N-methyl-3-phenylpropan-1-amine hydrochloride. The oxidative processes occurring in fluoxetine are pH-dependent and were ascribed to the chemical moieties present in the molecule: the secondary amine group and the substituted aromatic nucleus. To perform an unequivocal ascription, the structural preferences of the drug and metabolite were also determined, by Raman spectroscopy coupled to quantum mechanical calculations (at the DFT level). The analytical data obtained in this work will allow the development of a rapid and unequivocal spectroscopic procedure suitable for fluoxetine identification, as well as to distinguish between the drug and its main metabolite.