Conformational plasticity of DNA secondary structures: probing the conversion between i-motif and hairpin species by circular dichroism and ultraviolet resonance Raman spectroscopies.

The promoter regions of important oncogenes such as BCL2 and KRAS contain GC-rich sequences that can form distinctive noncanonical DNA structures involved in the regulation of transcription: G-quadruplexes on the G-rich strand and i-motifs on the C-rich strand. Interestingly, BCL2 and KRAS promoter i-motifs are highly dynamic in nature and exist in a pH-dependent equilibrium with hairpin and even with hybrid i-motif/hairpin species. Herein, the effects of pH and presence of cell-mimicking molecular crowding conditions on conformational equilibria of the BCL2 and KRAS i-motif-forming sequences were investigated by ultraviolet resonance Raman (UVRR) and circular dichroism (CD) spectroscopies. Multivariate analysis of CD data was essential to model the presence and identity of the species involved. Analysis of UVRR spectra measured as a function of pH, performed also by the two-dimensional correlation spectroscopy (2D-COS) technique, showed the role of several functional groups in the DNA conformational transitions, and provided structural and dynamic information. Thus, the UVRR investigation of intramolecular interactions and of local and environmental dynamics in promoting the different species induced by the solution conditions provided valuable insights into i-motif conformational transitions. The combined use of the two spectroscopic tools is emphasized by the relevant possibility of working in the same DNA concentration range and by the heterospectral UVRR/CD 2D-COS analysis. The results of this study shed light on the factors that can influence at the molecular level the equilibrium between the different conformational species putatively involved in the oncogene expression.

Ligand binding to G-quadruplex DNA: new insights from ultraviolet resonance Raman spectroscopy.

G-Quadruplexes (G4s) are noncanonical nucleic acid structures involved in the regulation of several biological processes of many organisms. The rational design of G4-targeting molecules developed as potential anticancer and antiviral therapeutics is a complex problem intrinsically due to the structural polymorphism of these peculiar DNA structures. The aim of the present work is to show how Ultraviolet Resonance Raman (UVRR) spectroscopy can complement other techniques in providing valuable information about ligand/G4 interactions in solution. Here, the binding of BRACO-19 and Pyridostatin - two of the most potent ligands - to selected biologically relevant G4s was investigated by polarized UVRR scattering at 266 nm. The results give new insights into the binding mode of these ligands to G4s having different sequences and topologies by performing an accurate analysis of peaks assigned to specific groups and their changes upon binding. Indeed, the UVRR data not only show that BRACO-19 and Pyridostatin interact with different G4 sites, but also shed light on the ligand and G4 chemical groups really involved in the interaction. In addition, UVRR results complemented by circular dichroism data clearly indicate that the binding mode of a ligand can also depend on the conformation(s) of the target G4. Overall, these findings demonstrate the utility of using UVRR spectroscopy in the investigation of G4s and G4-ligand interactions in solution.

Crowding and conformation interplay on human DNA G-quadruplex by ultraviolet resonant Raman scattering.

The G-quadruplex-forming telomeric sequence (TTAGGG)4TT was investigated by polarized Ultraviolet Resonance Raman Scattering (UVRR) at 266 nm. The presence of 40% poly(ethylene glycol) and the so-called "self-crowding" condition were used to induce the hybrid-to-parallel topology transition. Analysis of frequency shifts with temperature showed the role of several functional groups in the topological transitions and provides structural dynamical information. Circular dichroism under similar conditions was used as a reference. UVRR shed light on the effect of intramolecular interactions and of local and environmental dynamics in promoting different G-quadruplex topologies, induced by solution conditions or by temperature changes. Overall, these findings showed the enormous potential of this spectroscopy for G-quadruplex conformational studies.

Physico-chemical properties of aqueous drug solutions: From the basic thermodynamics to the advanced experimental and simulation results.

The physical chemical properties of aqueous solutions of model compounds are illustrated in relation to hydration and solubility issues by using three perspectives: thermodynamic, spectroscopic and molecular dynamics simulations. The thermodynamic survey of the fundamental backgrounds of concentration dependence and experimental solubility results show some peculiar behavior of aqueous solutions with several types of similar solutes. Secondly, the use of a variety of experimental spectroscopic devices, operating under different experimental conditions of dimension and frequency, has produced a large amount of structural and dynamic data on aqueous solutions showing the richness of the information produced, depending on where and how the experiment is carried out. Finally, the use of molecular dynamics computational work is presented to highlight how the different types of solute functional groups and surface topologies organize adjacent water molecules differently. The highly valuable contribution of computer simulation studies in providing molecular explanations for experimental deductions, either of a thermodynamic or spectroscopic nature, is shown to have changed the current knowledge of many aqueous solution processes. While this paper is intended to provide a collective view on the latest literature results, still the presentation aims at a tutorial explanation of the potentials of the three methodologies in the field of aqueous solutions of pharmaceutical molecules.

Molecular Dynamics and Neutron Scattering Studies of Mixed Solutions of Caffeine and Pyridine in Water.

Insight into the molecular interactions of homotactic and heterotactic association of caffeine and pyridine in aqueous solution is given on the basis of both experimental and simulation studies. Caffeine is about 5 times more soluble in a 3 m aqueous pyridine solution than it is in pure water (an increase from ∼0.1 m to 0.5 m). At this elevated concentration the system becomes suitable for neutron scattering study. Caffeine-pyridine interactions were studied by neutron scattering and molecular dynamics simulations, allowing a detailed characterization of the spatial and orientational structure of the solution. It was found that while pyridine-caffeine interactions are not as strong as caffeine-caffeine interactions, the pyridine-caffeine interactions still significantly disrupted caffeine-caffeine stacking. The alteration of the caffeine-caffeine stacking, occasioned by the presence of pyridine molecules in solution and the consequent formation of heterotactic interactions, leads to the experimentally detected increase in caffeine solubility.

Myelography Iodinated Contrast Media. 2. Conformational Versatility of Iopamidol in the Solid State.

The phenomenon of polymorphism is of great relevance in pharmaceutics, since different polymorphs have different physicochemical properties, e.g., solubility, hence, bioavailability. Coupling diffractometric and spectroscopic experiments with thermodynamic analysis and computational work opens to a methodological approach which provides information on both structure and dynamics in the solid as well as in solution. The present work reports on the conformational changes in crystalline iopamidol, which is characterized by atropisomerism, a phenomenon that influences both the solution properties and the distinct crystal phases. The conformation of iopamidol is discussed for three different crystal phases. In the anhydrous and monohydrate crystal forms, iopamidol molecules display a syn conformation of the long branches stemming out from the triiodobenzene ring, while in the pentahydrate phase the anti conformation is found. IR and Raman spectroscopic studies carried out on the three crystal forms, jointly with quantum chemical computations, revealed that the markedly different spectral features can be specifically attributed to the different molecular conformations. Our results on the conformational versatility of iopamidol in different crystalline phases, linking structural and spectroscopic evidence for the solution state and the solid forms, provide a definite protocol for grasping the signals that can be taken as conformational markers. This is the first step for understanding the crystallization mechanism occurring in supersaturated solution of iopamidol molecules.

Stacking and Branching in Self-Aggregation of Caffeine in Aqueous Solution: From the Supramolecular to Atomic Scale Clustering.

The dynamical and structural properties of caffeine solutions at the solubility limit have been investigated as a function of temperature by means of MD simulations, static and dynamic light scattering, and small angle neutron scattering experiments. A clear picture unambiguously supported by both experiment and simulation emerges: caffeine self-aggregation promotes the formation of two distinct types of clusters: linear aggregates of stacked molecules, formed by 2-14 caffeine molecules depending on the thermodynamic conditions and disordered branched aggregates with a size in the range 1000-3000 Å. While the first type of association is well-known to occur under room temperature conditions for both caffeine and other purine systems, such as nucleotides, the presence of the supramolecular aggregates has not been reported previously. MD simulations indicate that branched structures are formed by caffeine molecules in a T-shaped arrangement. An increase of the solubility limit (higher temperature but also higher concentration) broadens the distribution of cluster sizes, promoting the formation of stacked aggregates composed by a larger number of caffeine molecules. Surprisingly, the effect on the branched aggregates is rather limited. Their internal structure and size do not change considerably in the range of solubility limits investigated.

"The Good, the Bad and the Ugly" of Chitosans.

The objective of this paper is to emphasize the fact that while consistent interest has been paid to the industrial use of chitosan, minor attention has been devoted to spread the knowledge of a good characterization of its physico-chemical properties. Therefore, the paper attempts to critically comment on the conflicting experimental results, highlighting the facts, the myths and the controversies. The goal is to indicate how to take advantage of chitosan versatility, to learn how to manage its variability and show how to properly tackle some unexpected undesirable features. In the sections of the paper various issues that relate chitosan properties to some basic features and to advanced solutions and applications are presented. The introduction outlines some historical pioneering works, where the chemistry of chitosan was originally explored. Thereafter, particular reference is made to analytical purity, characterization and chain modifications. The macromolecular characterization is mostly related to molecular weight and to degree of acetylation, but also refers to the conformational and rheological properties and solution stability. Then, the antimicrobial activity of chitosan in relation with its solubility is reviewed. A section is dedicated to the formulation of chitosan biomaterials, from gel to nanobeads, exploring their innovative application as active carrier nanoparticles. Finally, the toxicity issue of chitosan as a polymer and as a constructed nanomaterial is briefly commented in the conclusions.

Myelography iodinated contrast media. I. Unraveling the atropisomerism properties in solution.

The present work reports a thorough conformational analysis of iodinated contrast media: iomeprol, iopamidol (the world's most utilized contrast agent), and iopromide. Its main aim is the understanding of the complex structural features of these atropisomeric molecules, characterized by the presence of many conformers with hindered rotations, and of the role of atropisomerism in the physicochemical properties of their aqueous solutions. The problem was tackled by using an extensive analysis of (13)C NMR data on the solutions of whole molecules and of simple precursors in addition to FT-IR investigation and molecular simulations. This analysis demonstrated that out of the many possible atropisomers, only a few are significantly populated, and their relative population is provided. The conformational analysis also indicated that the presence of a sterically hindered amidic bond, allowing a significant population of cis forms (E in iopromide and exo in iomeprol), may be the basis for an increased thermodynamic solubility of concentrated solutions of iomeprol.

The Interaction of Sorbitol with Caffeine in Aqueous Solution.

Molecular dynamics simulations were carried out on a system of caffeine interacting with the sugar alcohol sorbitol. The system examined had a caffeine concentration 0.083 m and a sugar concentration 1.08 m. The trajectories of all molecules in the system were collected over a period of 80 ns and analyzed to determine whether there is any tendency for sorbitol to bind to caffeine, and if so, by what mechanism. The results show that the sorbitol molecules have an affinity for the caffeine molecules and that the binding occurred by the interaction of the aliphatic hydrophobic protons of the sugar with the caffeine face. This intermolecular association via face-to-face stacking, as suggested by simulation studies, is similar to that found for sucrose and for D-glucose, which overwhelmingly exists in the pyranose ring chair form in aqueous solution, as well as for caffeine-caffeine association. The sorbitol molecules, however, exist as relatively extended chains and are, therefore, topologically quite different from the sugars sucrose and glucose. The comparison of the average conformation of sorbitol molecules bound to caffeine with that of molecules in the free state shows a substantial similarity.

Chitosan nanoparticles: preparation, size evolution and stability.

PURPOSE: Characterisation of chitosan-tripolyphosphate nanoparticles is presented with the aim of correlating particle shape and morphology, size distribution, surface chemistry, and production automatisation with preparation procedure, chitosan molecular weight and loaded protein. METHODS: Nanoparticles were prepared by adding drop wise a tripolyphosphate-pentasodium solution to chitosan solutions under stirring. Trehalose, mannitol and polyethylene-glycol as bioprotectants were used to prevent particle aggregation and to reduce mechanical stress during freezing and drying processes. RESULTS: As a novel result, time evolution of the particle size distribution curve showed the presence of a bimodal population composed of a fraction of small particles and of a second fraction of larger particles attributed to the rearrangement of particles after the addition of tripolyphosphate. Storage for 4 weeks resulted in a slight increase in average size, due to the continuous rearrangement of small particles. Improvement of nanoparticle stability after lyophilisation and spray-drying was observed in the presence of all bioprotectants. Trehalose was the best protectant for both methods. Finally, in vivo tests using chick embryos assessed the biocompatibility of chitosan, tripolyphosphate and the nanoparticles. CONCLUSION: The simple ionotropic gelation method with low-MW chitosan was effective in achieving reproducible nanoparticles with the desired physico-chemical and safety characteristics.

Caffeine and sugars interact in aqueous solutions: a simulation and NMR study.

Molecular dynamics simulations were carried out on several systems of caffeine interacting with simple sugars. These included a single caffeine molecule in a 3 m solution of α-D-glucopyranose, at a caffeine concentration of 0.083 m, a single caffeine in a 3 m solution of ß-D-glucopyranose, and a single caffeine molecule in a 1.08 m solution of sucrose (table sugar). Parallel nuclear magnetic resonance titration experiments were carried out on the same solutions under similar conditions. Consistent with previous thermodynamic experiments, the sugars were found to have an affinity for the caffeine molecules in both the simulations and experiments, and the binding in these complexes occurs by face-to-face stacking of the hydrophobic triad of protons of the pyranose rings against the caffeine face, rather than by hydrogen bonding. For the disaccharide, the binding occurs via stacking of the glucose ring against the caffeine, with a lesser affinity for the fructose observed. These findings are consistent with the association being driven by hydrophobic hydration and are similar to the previously observed binding of glucose rings to various other planar molecules, including indole, serotonin, and phenol.

Biophysical functionality in polysaccharides: from Lego-blocks to nano-particles.

The objective of the paper is to show the very important biophysical concepts that have been developed with polysaccharides. In particular, an attempt will be made to relate "a posteriori" the fundamental aspects, both experimental and theoretical, with some industrial applications of polysaccharide-based materials. The overview of chain conformational aspects includes relationships between topological features and local dynamics, exemplified for some naturally occurring carbohydrate polymers. Thus, by using simulation techniques and computational studies, the physicochemical properties of aqueous solutions of polysaccharides are interpreted. The relevance of conformational disorder-order transitions, chain aggregation, and phase separation to the underlying role of the ionic contribution to these processes is discussed. We stress the importance of combining information from analysis of experimental data with that from statistical-thermodynamic models for understanding the conformation, size, and functional stability of industrially important polysaccharides. The peculiar properties of polysaccharides in industrial applications are summarized for the particularly important example of nanoparticles production, a field of growing relevance and scientific interest.

Weakly hydrated surfaces and the binding interactions of small biological solutes.

Extended planar hydrophobic surfaces, such as are found in the side chains of the amino acids histidine, phenylalanine, tyrosine, and tryptophan, exhibit an affinity for the weakly hydrated faces of glucopyranose. In addition, molecular species such as these, including indole, caffeine, and imidazole, exhibit a weak tendency to pair together by hydrophobic stacking in aqueous solution. These interactions can be partially understood in terms of recent models for the hydration of extended hydrophobic faces and should provide insight into the architecture of sugar-binding sites in proteins.

Molecular dynamics simulation studies of caffeine aggregation in aqueous solution.

Molecular dynamics simulations were carried out on a system of eight independent caffeine molecules in a periodic box of water at 300 K, representing a solution near the solubility limit for caffeine at room temperature, using a newly developed CHARMM-type force field for caffeine in water. Simulations were also conducted for single caffeine molecules in water using two different water models (TIP3P and TIP4P). Water was found to structure in a complex fashion around the planar caffeine molecules, which was not sensitive to the water model used. As expected, extensive aggregation of the caffeine molecules was observed, with the molecules stacking their flat faces against one another like coins, with their methylene groups staggered to avoid steric clashes. A dynamic equilibrum was observed between large n-mers, including stacks with all eight solute molecules, and smaller clusters, with the calculated osmotic coefficient being in acceptable agreement with the experimental value. The insensitivity of the results to water model and the congruence with experimental thermodynamic data suggest that the observed stacking interactions are a realistic representation of the actual association mechanism in aqueous caffeine solutions.

Sugar-binding sites on the surface of the carbohydrate-binding module of CBH I from Trichoderma reesei.

Molecular dynamics simulations were carried out for a system consisting of the carbohydrate-binding module (CBM) of the cellulase CBH I from Trichoderma reesei (Hypocrea jecorina) in a concentrated solution of ß-D-glucopyranose, to determine whether there is any tendency for the sugar molecules to bind to the CBM. In spite of the general tendency of glucose to behave as an osmolyte, a marked tendency for the sugar molecules to bind to the protein was observed. However, the glucose molecules tended to bind only to specific sites on the protein. As expected, the hydrophobic face of the sugar molecules, comprising the axial H1, H3, and H5 aliphatic protons, tended to adhere to the flat faces of the three tyrosine side chains on the planar binding surface of the CBM. However, a significant tendency to bind to a groove-like feature on the upper surface of the CBM was also observed. These results would not be inconsistent with a model of the mechanism for this globular domain in which the cellodextrin chain being removed from the surface of crystalline cellulose passes over the upper surface of the CBM, presumably then available for hydrolysis in the active site tunnel of this processive cellulase.

Marine polysaccharides in microencapsulation and application to aquaculture: "from sea to sea".

This review's main objective is to discuss some physico-chemical features of polysaccharides as intrinsic determinants for the supramolecular structures that can efficiently provide encapsulation of drugs and other biological entities. Thus, the general characteristics of some basic polysaccharides are outlined in terms of their conformational, dynamic and thermodynamic properties. The analysis of some polysaccharide gelling properties is also provided, including the peculiarity of the charged polysaccharides. Then, the way the basic physical chemistry of polymer self-assembly is made in practice through the laboratory methods is highlighted. A description of the several literature procedures used to influence molecular interactions into the macroscopic goal of the encapsulation is given with an attempt at classification. Finally, a practical case study of specific interest, the use of marine polysaccharide matrices for encapsulation of vaccines in aquaculture, is reported.

Trehalose amorphization and recrystallization.

The stability of the amorphous trehalose prepared by using several procedures is presented and discussed. Amorphization is shown to occur by melting (T(m)=215 degrees C) or milling (room temperature) the crystalline anhydrous form TRE-beta. Fast dehydration of the di-hydrate crystalline polymorph, TRE-h, also produces an amorphous phase. Other dehydration procedures of TRE-h, such as microwave treatment, supercritical extraction or gentle heating at low scan rates, give variable fractions of the polymorph TRE-alpha, that undergo amorphization upon melting (at lower temperature, T(m)=130 degrees C). Additional procedures for amorphization, such as freeze-drying, spray-drying or evaporation of trehalose solutions, are discussed. All these procedures are classified depending on the capability of the undercooled liquid phase to undergo cold crystallization upon heating the glassy state at temperatures above the glass transition temperature (T(g)=120 degrees C). The recrystallizable amorphous phase is invariably obtained by the melt of the polymorph TRE-alpha, while other procedures always give an amorphous phase that is unable to crystallize above T(g). The existence of two different categories is analyzed in terms of the transformation paths and the hypothesis that the systems may exhibit different molecular mobilities.

Tuning supramolecular structuring at the nanoscale level: nonstoichiometric soluble complexes in dilute mixed solutions of alginate and lactose-modified chitosan (chitlac).

Two oppositely charged polysaccharides, alginate and a lactose-modified chitosan (chitlac), have been used to prepare dilute binary polymer mixtures at physiological pH (7.4). Because of the negative charge on the former polysaccharide and the positive charge on the latter, polyanion-polycation complex formation occurred. A complete miscibility between the two polysaccharides was attained in the presence of both high (0.15 M) and low (0.015 M) concentrations of simple 1:1 supporting salt (NaCl), as confirmed by turbidity measurements; phase separation occurred for intermediate values of the ionic strength (I). The binary solutions were further characterized by means of light scattering, specific viscosity, and fluorescence quenching measurements. All of these techniques pointed out the fundamental role of the electrostatic interactions between the two oppositely charged polysaccharides in the formation of nonstoichiometric polyelectrolyte soluble complexes in dilute solution. Fluorescence depolarization (P) experiments showed that the alginate chain rotational mobility was impaired by the presence of the cationic polysaccharide when 0.015 M NaCl was used. Moreover, upon addition of calcium, the P values of the binary polymer mixture in 0.015 M NaCl increased more rapidly than that of an alginate solution without chitlac, suggesting an efficient crowding of the negatively charged alginate chains caused by the polycation.

Exploring conformational energy landscape of glassy disaccharides by cross polarization magic angle spinning 13C NMR and numerical simulations. I. Methodological aspects.

This article demonstrates the ability of chemical shift surfaces to provide information on distributions of various conformations of disaccharides in the glassy, solid state. The validity of the general method leading to a simulation of inhomogeneous (13)C chemical shift distributions is discussed in detail. In particular, a proper consideration of extrema and saddle points of the chemical shift map correctly accounts for the observed discontinuities in the experimental cross polarization magic angle spinning spectra. Provided that these basic requirements are met, density functional theory/gauge-independent atomic orbital chemical shift maps calculated on relaxed conformations lead to a very satisfactory description of the experimental line shapes. Using amorphous trehalose as a model disaccharide, the simulation unambiguously defines the range of most populated conformations in the glass.