Carla Cruz

Amino acids–nucleotides biomolecular recognition: from biological occurrence to affinity chromatography

In this review, the protein–DNA interactions are discussed considering different perspectives, and the biological occurrence of this interaction is explained at atomic level. The evaluation of the amino acid–nucleotide recognition has been investigated analysing datasets for predicting the association preferences and the geometry that favours the interaction.

Design of Protonated Polyazamacrocycles Based on Phenanthroline Motifs for Selective Uptake of Aromatic Carboxylate Anions and Herbicides

Three novel large polyazamacrocycles containing two 1,10-phenanthroline units connected by different polyamine spacers have been synthesised and their protonated forms used as receptors for several aromatic carboxylate anions. The receptors bind to the anions in a 1:1 stoichiometry and exhibit remarkable binding selectivity (see figure). Analysis shows that molecular recognition is governed by pi-pi stacking interactions and multiple N--HO==C hydrogen bonds.Three novel large polyazamacrocycles containing two 1,10-phenanthroline (phen) units connected by two polyamine spacers of different length, [32]phen(2)N(4), [30]phen(2)N(6) and Me(2)[34]phen(2)N(6), have been synthesised and their protonated forms used as receptors for binding studies with several aromatic carboxylate anions (benzoate (bzc(-)), 1-naphthalate (naphc(-)), 9-anthracenate (anthc(-)), pyrene-1-carboxylate (pyrc(-)), phthalate, (ph(2-)), isophthalate (iph(2-)), terephthalate (tph(2-)), 2,5-dihydroxy-1,4-benzenediacetate (dihyac(2-)) and, 1,3,5-benzenetricarboxylate (btc(3-))) and three herbicides (4-amino-3,5,6-trichloropyridine-2-carboxylate (ATCP(-)), dichlorophenoxyacetate (2,4-D(-)) and glyphosate (PMG(2-))) in water solution. The [30]phen(2)N(6) receptor was found to be the most suitable for binding the anions considered in a 1:1 stoichiometry. The three receptors exhibit a remarkable binding selectivity towards the extended aromatic anion pyrc(-) at low pH values. Their binding affinities for the monocarboxylate anions decrease with the extension of the aromatic system in the order pyrc(-)>anthc(-)>naphc(-)>bzc(-), which indicates the presence of pi-pi stacking interactions in the molecular recognition of these anions. Molecular dynamics simulations carried out for the binding of {H(4)[30]phen(2)N(6)}(4+) and {H(6)Me(2)[34]phen(2)N(6)}(6+) with pyrc(-), anthc(-), naphc(-), iph(2-) and btc(3-) in water showed that these receptors adopt a folded conformation with the anion inserted between the two phen heads and that the molecular recognition is governed by pi-pi stacking interactions and multiple N--HO==C hydrogen bonds. The binding free energies estimated theoretically are very similar to those found by potentiometric methods, which supports the proposed binding arrangement.

Dinuclear copper and zinc complexes of a hexaazamacrocycle containing p-xylyl spacers and bridging anions: theoretical and spectroscopic studies

The hexaazamacrocycle 7,22-dimethyl-3,7,11,18,22,26-hexaazatricyclo[26.2.2.213,16]tetratriaconta-1(30),13,15,28,31,33-hexaene (Me2[30]pbz2N6) was synthesized and characterised by single crystal X-ray diffraction. The macrocycle adopts a conformation with the two aromatic rings almost parallel at a distance of ca. 4.24 A, but displaced relative to each other by ca. 1.51 A. The protonation constants of this compound and the stability constants of its complexes with Cu2+ and Zn2+, were determined in water–methanol (9 ∶ 1 v/v) at 25 °C with ionic strength 0.10 mol dm−3 in KCl. The potentiometric and spectroscopic studies (NMR of zinc, cadmium and lead complexes, and EPR of the copper complexes) indicate the formation of only dinuclear complexes. The association constants of the dinuclear copper complex with anions (thiocyanate, terephthalate and glyphosate) and neutral molecules (1,4-benzenedimethanol, p-xylylenediamine and terephthalic acid) were determined at 20 °C in methanol. The structural preferences of this ligand and of its dinuclear copper(II) complex with a variety of bridging ligands were evaluated theoretically by molecular mechanics calculations (MM) and molecular dynamics (MD) using quenching techniques.

Screening nucleotide binding to amino acid-coated supports by surface plasmon resonance and nuclear magnetic resonance

Here, we describe a rapid and efficient screening method using surface plasmon resonance (SPR) and saturation transfer difference–nuclear magnetic resonance (STD-NMR) spectroscopy to yield information regarding the residues involved in nucleotide binding to amino acid-coated supports. The aim of this work was to explore the use of these spectroscopic techniques to study amino acid–nucleotide interactions in order to improve the binding specificity of the amino acid ligands used to purify plasmid DNA. For SPR, we present a strategy that immobilizes arginine and lysine on a surface as model supports, and we analyze binding responses when synthetic homo-deoxyoligonucleotides are injected over the amino acid surface. The binding responses are detectable and reproducible despite the small size of the immobilized amino acids. Using STD-NMR, we performed epitope mapping of homo-deoxyoligonucleotides bound to l-arginine–bisoxyran–Sepharose and l-lysine–Sepharose supports. Polynucleotide binding preferences differed; for example, polyC interacted preferentially through its backbone with the two supports, whereas polyT bound the supports through its thymine moiety. STD-NMR combined with SPR measurements was successfully used to screen amino acid–nucleotide interactions and determine the binding affinities of the complexes.

Targeting of mitochondria-endoplasmic reticulum by fluorescent macrocyclic compounds.

Background Useful probes of the intracellular environment that target a specific organelle in order to allow direct observation of the changes in these regions is of high current interest. Macrocyclic ligands have already revealed themselves as important selective hosts in some biological applications, forming stable and specific complexes. Therefore, in this paper, several macrocyclic ligands are evaluated as potential molecular probes. Methodology Four polyammonium macrocycles and one macrotricyclic bearing pyridine and phenanthroline chromophores have been synthesised and evaluated as molecular probes. The cytotoxicity of the compounds has been analyzed using human breast cancer cells (MCF-7), non-cancerous human dermal fibroblasts (NHDF) and human adult dermal skin fibroblasts from a breast cancer patient (P14). All the compounds showed low toxicity at concentrations ranging from 10 nM to 10 µM, except for [32]phen2N4 which proved to be highly cytotoxic for MCF-7 cells. Flow cytometry studies evidenced that the percentage of apoptotic and necrotic MCF-7 and NHDF cells induced by the compounds is considerably low. Also, flow cytometry analysis showed that some compounds seem to modify the mitochondrial membrane potential (MMP) of the cells. Fluorescence microscopy evidenced that compounds easily cross the plasma membrane (5 min) and accumulated into the mitochondria, as confirmed by co-localization with MitoTracker Green™. The fluorescence images also evidenced an intact mitochondria structure after 48 h. Moreover, reticular staining suggestive of endoplasmic reticulum (ER) localization, in addition to the mitochondrial one, has been found by confocal microscopy. Conclusion Our study reveals that compounds Me2[28]py2N6, cryptphen, [16]phenN2, [30]phen2N6, have low toxicity and localize in mitochondria and ER. The ability of these compounds for translocating the cellular membrane (5 min) without special conditioning of the cells or derivatization of the probe, the time-dependent localization (48 h) and the cellular viability provide a proof-of-concept towards their use as promising probes towards biomedical studies.

Sensitive detection of peptide-minicircle DNA interactions by surface plasmon resonance.

Minicircle DNA (mcDNA) is recently becoming an exciting source of genetic material for therapeutic purposes due to its exceptional biocompatibility and efficiency over typical DNA. However, its widespread use is yet restrained because of the absence of an efficient technology that allows its purification. Here, the precise conditions of mcDNA interaction with novel arginine-arginine dipeptide ligands were explored to promote binding and recovery of these biopharmaceuticals. Such interactions were investigated by taking advantage of a highly sensitive method based on surface plasmon resonance (SPR) to screen, in real-time, for ligand-coupled biomolecules, while preserving mcDNA integrity. Through this analytic approach, we detected dynamic binding responses that are dependent on buffer type, mcDNA electrokinetic potential, and temperature conditions. Remarkably, the results obtained revealed that the ligands possess high affinity to mcDNA molecules under low salt buffers, and low affinity in the presence of salt, suggesting that electrostatic interactions mainly govern ligand-analyte coupling. These findings provide important insights for an active manipulation of parameters that promote mcDNA recovery and purification. Above all, this study showed the crucial importance of SPR for future screening of other ligands that, like the one described herein, can be used to design mcDNA recovery platforms which will have significant impact in biopharmaceutical-based therapeutics.

Affinity analysis between immobilized L-arginine and plasmid isoforms provided by surface plasmon resonance

A strategy for studying the binding mode of plasmid isoforms to an immobilized amino acid as a model support has been developed using Surface Plasmon Resonance (SPR). The binding of L-arginine to plasmid isoforms of pGL101, pUC19 and pVAX1-LacZ (supercoiled, open-circular and linear) is examined by measuring the equilibrium dissociation constants (KD). L-Arginine was immobilized on a CM5 sensor chip and bound to plasmid isoforms with similar SPR binding profiles (square-shaped) and binding affinities ranging between 10−4 and 10−9 M. There are significant differences in the apparent affinity that is correlated with the three plasmids (2.39, 2.69 and 6.05 kbp). pGL101 shows the highest binding on the arginine surface followed by pVAX1-LacZ, while pUC19 shows the lowest binding. For the three plasmid isoforms, the supercoiled ones have the higher binding affinity to the arginine surface. Different buffer environments affect the interaction strength with an increase in response for Tris–HCl and a marked decrease for high salt concentrations. The knowledge of the affinity parameters is expected to provide further insights into the effect of plasmid topology on the purification by affinity chromatography.

Metal complexes of a dipyridine octaazamacrocycle: stability constants, structural and modelling studies

Two 28-membered octaazamacrocycles, [28]py2N6 and Me2[28]py2N6, have been synthesized. The protonation constants of the N-methyl derivative and the stability constants of its complexes with Ni2+, Cu2+, Zn2+, Cd2+, and Pb2+ were determined at 25 °C in 0.10 mol dm−3 KNO3. The high overall basicity of Me2[28]py2N6 is ascribed to the weaker repulsion between protonated contiguous charged ammonium sites separated by propyl chains. These studies together with NMR, UV-vis and EPR spectroscopies indicated the presence of mono- and di-nuclear species. The single crystal structure of the complex [Ni2([28]py2N6)(H2O)4]Cl4·3H2O was determined, and showed each nickel centre in a distorted octahedral co-ordination environment. The nickel centres are held within the macrocycle at a large distance of 6.991(8) A from each other. The formation of mononuclear complexes was evaluated theoretically via molecular mechanics (MM) and molecular dynamics (MD) calculations and showed that these large macrocycles have sufficient flexibility to encapsulate metal ions with different stereo-electronic sizes. Structures for small and large metal ions are proposed.

Polyazamacrocycles as Potential Antitumor Agents for Human Prostate Cancer Cells

Polyazamacrocycles are currently being studied and used in a variety of applications beyond their traditional place in supramolecular and co‐ordination chemistry. This study suggests additional applications of these compounds with particular emphasis on their use as antiproliferative agents that could be potentially used to treat cancer. Four polyazamacrocycles were tested in human prostate cancer LNCaP and prostate epithelial PNTA1 cells to analyze changes in cell proliferation and cell death capabilities. Their intracellular localization was also evaluated by confocal microscopy. The results show a decrease in proliferation rate and cell viability of LNCaP and PNTA1, after treatment with these compounds. The decrease in the number of viable cells is similar for the majority of the compounds studied, and at higher concentration, the proliferation efficiency decreased significantly in the cell lines studied. Also, our results suggest that L and L2 induce early apoptosis in PNTA1 cells and late apoptosis/necrosis in LNCaP cells. The compounds did not induce a significant increase in necrosis of both cell types. Although the compounds did not localize in a unique organelle, all of them have as main target the Golgi apparatus and other localization profiles differed depending on the cell line.

On pseudo-complex bases for monogenic polynomials

In the recent past, the problem of constructing bases for spaces of monogenic polynomials, in the framework of Clifford Analysis, has been considered by several authors, using different methods. In this talk we consider bases of 3D monogenic polynomials isomorphic to the complex powers, which are particularly easy to handle, from the computational point of view. Explicit constructions of such polynomial bases are performed and a numerical cost comparison with the well known Fueter polynomial basis is carried out.