Publications

Abstract The influence of the heteroatoms in the conformational, energetic, and reactivity trends exhibited by morpholine and thiomorpholine isosteres was obtained from computational and experimental thermochemical studies. For those purposes, the gas-phase standard (p = 0.1 MPa) molar enthalpies of formation of the compounds, at T = 298.15 K, were determined from the experimental values of the standard molar. enthalpies of formation, in the liquid phase, and of the standard molar: enthalpies of vaporization, obtained by calorimetric techniques, and also from composite G3(MP2)// B3LYP calculations making use of appropriate working reactions. A very good agreement was found between the calculated and the experimental gas-phase enthalpies of formation. The computational study was further extended to the calculation of other gas-phase thermodynamic properties these compounds, namely, the N-H or C-H bond dissociation enthalpies, gas-phase acidities and basicitics, proton affinities and adiabatic ionization enthalpies, and the energies and structures of the conformational stereoisomers of morpholine and thiomorpholine.

Abstract Firefly bioluminescence presents a light emitting profile with a form of a flash, due to the firefly luciferase-catalyzed formation of inhibitory products. These impair the binding of the substrate luciferin to the active site of the enzyme. However, this luciferase catalyzed pathways may not be the only ones responsible for the flash profile. The oxidation and isomerisation of the substrate luciferin lead to the formation of compounds that are also known inhibitors of firefly bioluminescence. So, the objective of this Letter was to analyze if these reactions could be capable of interfering with the bioluminescence reaction.

Abstract Pollen of Betula pendula, Ostrya carpinifolia and Carpinus betulus was exposed in vitro to two levels of NO2 (about 0.034 and 0.067 ppm) both below current atmospheric hour-limit value acceptable for human health protection in Europe (0.11 ppm for NO2). Experiments were performed under artificial solar light with temperature and relative humidity continuously monitored. The viability, germination and total soluble proteins of all the pollen samples exposed to NO2 decreased significantly when compared with the non-exposed. The polypeptide profiles of all the pollen samples showed bands between 15 and 70 kDa and the exposure to NO2 did not produce any detectable changes in these profiles. However, the immunodetection assays indicated higher IgE recognition by patient sera sensitized to the pollen extracts from all exposed samples in comparison to the non-exposed samples. The common reactive bands to the three pollen samples correspond to 58 and 17 kDa proteins.

Abstract This paper is concerned with computational and experimental thermochemical studies of azepan and azepan-1-ylacetonitrile, molecules whose flexible ring structure provides several conformational forms with low energy barriers among them. The computational study describes the energetic analysis of the six most stable conformers on the potential energy surfaces and the determination of their gas-phase standard enthalpy of formation at the reference temperature of 298.15 K. The same gas-phase enthalpic parameters are also derived from the enthalpies of formation in the liquid phase and the enthalpies of vaporization, at T = 298.15 K, determined experimentally using the combustion calorimetry and the Calvet microcalorimetry techniques, respectively. The experimental data reported in this work for the two titled compounds together with other available in the literature for related molecules enabled the establishment of an increments scheme, providing a reliable approach on the prevision of gas-phase enthalpy of formation of cyclic/acyclic hydrocarbons and amines. Complementary, natural bond orbital (NBO) calculations were also performed, allowing an advance on the analysis of the structural and reactivity behavior of these type of compounds.

Abstract A series hydroxycinnamic and gallic acids and their derivatives were studied with the aim of evaluating their in vitro antioxidant properties both in homogeneous and in cellular systems. It was concluded from the oxygen radical absorbance capacity-fluorescein (ORAC-FL), 1,1-diphenyl-2-picrylhydrazyl (DPPH), and cyclic voltammetry data that some compounds exhibit remarkable antioxidant properties. In general, in homogeneous media (DPPH assay), galloyl-based cinnamic and benzoic systems (compounds 7-11) were the most active, exhibiting the lowest oxidation potentials in both dimethyl sulfoxide (DMSO) and phosphate buffer. Yet, p-coumaric acid and its derivatives (compounds 1-3) disclosed the highest scavenging activity toward peroxyl radicals (ORAC-FL assay). Interesting structure-property-activity relationships between ORAC-FL, or DPPH radical, and redox potentials have been attained, showing that the latter parameter can be a valuable antioxidant measure. It was evidenced that redox potentials are related to the structural features of cinnamic and benzoic systems and that their activities are also dependent on the radical generated in the assay. Electron spin resonance data of the phenoxyl radicals generated both in DMSO and phosphate buffer support the assumption that radical stability is related to the type of phenolic system. Galloyl-based cinnamic and benzoic ester-type systems (compounds 9 and 11) were the most active and effective compounds in cell-based assays (51.13. 1.27% and 54.90. 3.65%, respectively). In cellular systems, hydroxycinnamic and hydroxybenzoic systems operate based on their intrinsic antioxidant outline and lipophilic properties, so the balance between these two properties is considered of the utmost importance to ensure their performance in the prevention or minimization of the effects due to free radical overproduction.

Abstract DHQ2 (type II dehydroquinase), which is an essential enzyme in Helicobacter pylori and Mycobacterium tuberculosis and does not have any counterpart in humans, is recognized to be an attractive target for the development of new antibacterial agents. Computational and biochemical studies that help understand in atomic detail the catalytic mechanism of these bacterial enzymes are reported in the present paper. A previously unknown key role of certain conserved residues of these enzymes, as well as the structural changes responsible for triggering the release of the product from the active site, were identified. Asp89*/Asp88* from a neighbouring enzyme subunit proved to be the residue responsible for the deprotonation of the essential tyrosine to afford the catalytic tyrosinate, which triggers the enzymatic process. The essentiality of this residue is supported by results from site-directed mutagenesis. For H. pylori DHQ2, this reaction takes place through the assistance of a water molecule, whereas for M. tuberculosis DHQ2, the tyrosine is directly deprotonated by the aspartate residue. The participation of a water molecule in this deprotonation reaction is supported by solvent isotope effects and proton inventory studies. MD simulation studies provide details of the required motions for the catalytic turnover, which provides a complete overview of the catalytic cycle. The product is expelled from the active site by the essential arginine residue and after a large conformational change of a loop containing two conserved arginine residues (Arg109/Arg108 and Arg113/Arg112), which reveals a previously unknown key role for these residues. The present study highlights the key role of the aspartate residue whose blockage could be useful in the rational design of inhibitors and the mechanistic differences between both enzymes. © The Authors Journal compilation © 2014 Biochemical Society.

Abstract The present work deals with the study of the anthocyanin profile of two different black carrots (Daucus carota L. ssp. sativus var. atrorubens Alef.) cultivars, associated with Antonina and Purple Haze varieties, from Cuevas Bajas (Malaga, Spain) and some of their antioxidant features. The main anthocyanins detected by LC-MS were found to correspond to five cyanidin-based anthocyanins: cyanidin 3-xylosylglucosylgalactoside, cyanidin 3-xylosylgalactoside and the sinapic, ferulic and coumaric acids derivative of cyanidin 3-xylosylglucosylgalactoside. The anthocyanins present in the black carrots were essentially acylated and their levels were found to correspond to 25% and 50% of the total phenolic content for the Purple Haze and Antonina varieties, respectively. Moreover, the reducing capacity of the two black carrots extracts (86.4 +/- 8.0 and 182.0 +/- 27 mu M TE/100 g fw) and the radical scavenging ability (17.6 +/- 9.0 and 240.0 +/- 54.0 mu M TE/100 g fw) expressed in Trolox equivalents units were determined. The antioxidant features of the black carrot extracts were shown to be significantly higher than those of orange carrots used herein for comparison. Overall, this work highlights the Cuevas Bajas black carrots as rich sources of anthocyanins with significant antioxidant capacities and good nutritional value.

Abstract Structural and computational studies to explore the WAT1 binding pocket in the structure-based design of inhibitors against the type II dehydroquinase (DHQ2) enzyme are reported. The crystal structures of DHQ2 from M. tuberculosis in complex with four of the reported compounds are described. The electrostatic interaction observed between the guanidinium group of the essential arginine and the carboxylate group of one of the inhibitors in the reported crystal structures supports the recently suggested role of this arginine as the residue that triggers the release of the product from the active site. The results of the structural and molecular dynamics simulation studies revealed that the inhibitory potency is favored by promoting interactions with WAT1 and the residues located within this pocket and, more importantly, by avoiding situations where the ligands occupy the WAT1 binding pocket. The new insights can be used to advantage in the structure-based design of inhibitors. © 2014 American Chemical Society.

Abstract Firefly oxyluciferin is a prime example in which pi-pi stacking interactions play an important role, by being the basis for the formation of sandwich-like oxyluciferin complexes. In the present study, we have used a theoretical methodology to further understand the effect of pi-pi stacking interactions in the properties of oxyluciferin molecule. More specifically, we have analysed the effect of resonance changes in oxyluciferin pi-pi stacking dimers. We have found that resonance changes have little effect on the ground state properties of the dimmers. More interestingly, by modulating the resonance of the dimmers we can obtain different transition energies and efficiencies. This results in changes in the degree of contributions made by the different orbital excitations that compose the excitation transition.

Abstract Structural, biochemical and computational studies to study substrate binding and the role of the conserved residues of the DHQ1 (type I dehydroquinase) enzyme active site are reported in the present paper. The crystal structure of DHQ1 from Salmonella typhi in complexwith (2R)-2-methyl-3-dehydroquinic acid, a substrate analogue, was solved at 1.5 Å. The present study reveals a previously unknown key role for conserved Glu46, Phe145 and Met205 and Gln236, Pro234 and Ala233 residues, with the latter three being located in the flexible substrate-covering loop. Gln236 was shown to be responsible for the folding of this loop and for the dramatic reduction of its flexibility, which triggers active site closure. Glu46 was found to be key in bringing the substrate close to the lysine/histidine catalytic pocket to initiate catalysis. The present study could be useful in the rational design of inhibitors of this challenging and recognized target for the development of novel herbicides and antimicrobial agents. © 2014 Biochemical Society.