New insights into the ultrafast photophysics of oxidized and reduced FAD in solution

The ultrafast photophysics of oxidized and reduced flavin adenine dinucleotide (FAD) in aqueous solution was studied by broadband UV-vis femtosecond transient absorption spectroscopy. We observed that oxidized FAD (FAD(ox)) in solution readily aggregates at submillimolar concentration. Upon excitation of FAD(ox), three excited-state lifetimes were found and assigned to three different species: the closed (stacked) conformation of the monomer (～5.4 ps), the open (extended) conformation of the monomer (～2.8 ns), and the dimer (～27 ps). In the case of the stacked conformation of the monomer, we show that intramolecular electron transfer from the adenine to the isoalloxazine ring occurs with a time constant of 5.4 ps and is followed by charge recombination on a faster time scale, namely, 390 fs. We additionally demonstrate that deprotonated reduced flavin (FADH(-)) undergoes biphotonic ionization under high excitation fluence and dissociates into a hydrated electron and the neutral semiquinone radical FADH(?).     

New insights into the interactions between dendrimers and surfactants by two dimensional NOE NMR spectroscopy

The interactions between polyamidoamine dendrimers and different surfactants including sodium dodecyl sulfate (SDS) and dodecyltrimethyl ammonium bromide in aqueous solutions have been investigated by a NOESY NMR technique. Strong NOE cross-peaks between hydrophobic chain protons of SDS and methylene protons of cationic dendrimers were found, suggesting a strong tendency for the long hydrophobic tails of SDS to associate with the hydrophobic pockets of dendrimers. The hydrophilic head of SDS localizes near the core or the boundary of each generation of dendrimers, and the hydrophobic chain of SDS localizes in the relative nonpolar pockets of dendrimers. The encapsulation of surfactant monomers by dendrimers is dependent on the charge type of the surfactants, the surface functionality, and the generation of dendrimers. The NOESY analysis provides a new insight into interactions between dendrimers and surfactants in comparison with previous investigations.     

New insights into urea action on proteins: a SANS study of the lysozyme case

We present a study on lysozyme dissolved in mixtures of water and urea, which is ubiquitously used as a protein denaturant. Despite the wide use of urea, the basic molecular mechanisms inducing protein unfolding are not still clarified. Small-angle neutron scattering (SANS) experiments have been performed using little amounts of denaturant in solutions in order to investigate the urea effect on lysozyme preceding the unfolding process. A global fit strategy, applied to analyze SANS experiments, provides an estimation of the average composition of the solvent in the close vicinity of the protein surface and the change of the protein-protein interactions due to the presence of urea. In particular, the thermodynamic equilibrium constant responsible for cosolvent balancing between the bulk and solvation layer has been determined. It turns out that urea is preferentially driven to the protein surface, confirming literature results at infinite dilute conditions. SANS data also reveal a possible variation of the protein net charge as a function of urea concentration, opening new perspectives and questions about the protein surface architecture at the first stages of unfolding processes.     

New insights into lung surfactant monolayers using vibrational sum frequency generation spectroscopy

At the air-water interface, interfacial molecular structure, intermolecular interactions, film relaxation and film respreading of model lung surfactant monolayers were studied using vibrational sum frequency generation (VSFG) spectroscopy combined with a Langmuir film balance. Chain-perdeuterated dipalmitoylphosphatidylcholine (DPPC-d62), palmitoyloleoyl-phosphatidylglycerol (POPG), palmitic acid (PA) and tripalmitin were investigated. In the DPPC-d62-PA binary monolayer, PA showed a condensing effect on the DPPC chains. On the contrary, in the DPPC-d62-POPG binary monolayer, POPG showed a fluidizing effect on the DPPC chains. In the ternary monolayer system of DPPC-d62-POPG-PA, the balance between the fluidizing and the condensing effect was also observed. In addition, the film relaxation behavior of DPPC-d62 and the enhanced film stability of DPPC-d62 caused by the addition of tripalmitin were observed. Real-time VSFG was also employed to study the respreading properties of a complex lung surfactant mixture containing DPPC-d62, POPG, PA and KL4 (a mimic of SP-B) peptide, which revealed DPPC enrichment after film compression.     

Probing redox proteins on a gold surface by single molecule fluorescence spectroscopy

The interaction between the fluorescently labeled redox protein, azurin, and a thin gold film is characterized using single-molecule fluorescence intensity and lifetime measurements. Fluorescence quenching starts at distances below 2.3 nm from the gold surface. At shorter distances the quantum yield may decrease down to fourfold for direct attachment of the protein to bare gold. Outside of the quenching range, up to fivefold enhancement of the fluorescence is observed on average with increasing roughness of the gold layer. Fluorescence-detected redox activity of individual azurin molecules, with a lifetime switching ratio of 0.4, is demonstrated for the first time close to a gold surface.     

New insights into the multiple minima problem

Minima distribution of thorough conformational searches of three peptides of different length ranging from five to nine residues, were compared with the density of states of a flexible molecule derived from the rotational isomeric approximation. It is observed that minima distributions generated from the conformational searches exhibit the same characteristics as the density of states derived from the rotational isomeric model: an asymmetric distribution with a maximum. These results together with a more profound understanding of the characteristics of the energy landscapes of polypeptides, provide new insights into the multiple minima problem. The implications in devising more robust conformational search strategies are discussed.     

New insights into the solubilization of Bodipy dyes

Several Bodipy dyes were synthesized with various substituents designed to improve the water solubility. Among the different synthetic strategies protection of sulfonate groups by pyrrole of indopyrrole appears efficient but the deprotection step does not offer viable routes. Conversion of the bromopyrene or iodo Bodipy compounds to sulfobetaine derivatives is feasible either by cross-coupling directly the ethynylsulfobetaine or by first cross-coupling 1-(N,N-dimethylamino)-prop-2-yne followed by quaternization of the dimethylamino residue with 1,3-propanesultone. Some of these dyes (Bodipy’s and pyrene) are reasonably soluble in water and remain highly fluorescent in polar solvents and water.     

New insights into the structure of polyelectrolyte complexes

The formation of polyelectrolyte complexes (PECs) from oppositely charged linear polyelectrolytes (PELs) was studied using static light scattering at various salt concentrations. The PELs used were poly(allylamine hydro chloride) (PAH) and the two polyanions poly(acrylic acid) (PAA) and poly(methacrylic acid) (PMAA). Physical characteristics such as the radii of gyration, molecular weights, and water contents of the PECs were determined at various molar mixing ratios. Despite relatively small differences in chemical structure between PAA and PMAA, fairly large differences were detected in these physical characteristics. Generally, PECs comprising PMAA were larger and contained more water. Moreover, by using cryogenic transmission electron microscopy, transmission microscopy and atomic force microscopy, shape and structure of the prepared PECs were investigated both in solution and after drying. The PECs were found to be spherical in solution and the shape was retained after freeze-drying. PECs adsorbed on silica surfaces and dried in air at room-temperature still showed a three-dimensional structure. However, the relatively low aspect ratios indicated that the PECs collapsed significantly due to interactions with the silica during adsorption and drying. At intermediate ionic strengths (1-10 mM), stagnation point adsorption reflectometry (SPAR) showed that the adsorption of low charged cationic PAH-PAA PECs on silica surfaces increased if the pH value was increased from pH 5.5 to 7.5.     

New insights into the mechanism of asymmetric hydrogenation catalysed by monophosphonite-rhodium complexes

The solvento complex [Rh(L)2(S)2]+ where L=tBuP(R-binaphthoxo) is shown to be in equilibrium with an eta-arene dirhodium complex and only weak, monodentate binding of alkenes is observed; in addition, an intermediate Rh alkyl hydride complex containing two coordinated monophosphonites is unambiguously characterised by NMR.     

Squaraines as reporter units: insights into their photophysics, protonation, and metal-ion coordination behaviour

The synthesis, photophysical properties, protonation, and metal-ion coordination features of a family of nine aniline-based symmetrical squaraine derivatives are reported. The squaraine scaffold displays very attractive photophysical properties for a signalling unit. These dyes show absorption and weakly Stokes-shifted, mirror-image-shaped emission bands in the visible spectral range and there are no hints of multiple emission bands. The mono-exponential fluorescence decay kinetics observed for all the derivatives indicate that only one excited state is involved in the emission. These data stress the interpretation that squaraines can be regarded as polymethine-type dyes. From a coordination chemistry point of view, the squaraines possess four potential binding sites; that is, two nitrogen atoms from the anilino groups and two oxygen atoms from the central C(4)O(2) four-membered ring. These coordination sites are part of a cross-conjugated pi-system and coordination events with protons or certain metal ions affect the electronic properties of the delocalised pi-system dramatically, resulting in a rich modulation of the colour of the squaraines. The absorption band at around 640 nm is blue-shifted when coordination at the anilino nitrogen atoms occurs, whereas coordination to the C(2)O(4) oxygen atoms results in the development of red-shifted bands. Addition of more than one equivalent of protons or metal cations could additionally entail mixed N,O- or N,N-coordinated complexes, manifested in the development of a broad band at 480 nm or complete bleaching in the visible range, respectively. Analysis of the spectrophotometric titration data with HYPERQUAD yielded the macroscopic and microscopic stability constants of the complexes. Theoretical modelling of the various protonated species by molecular mechanics methods and consideration of some of the title dyes within the framework of molecular chemosensing and molecular-scale "logic gates" complement this contribution.     

Linkers and catalysts immobilized on oxide supports: New insights by solid-state NMR spectroscopy

This review article describes classical and modern solid-state NMR methods that allow to gain insight into catalyst systems where one or two metal complexes are bound to oxide supports via bifunctional phosphine linkers, such as (EtO)₃Si(CH₂)₃PPh₂. Many aspects of the immobilized molecular catalysts can be elucidated with the corresponding NMR technique. The bulk of the support can be studied, as well as the interface of the support with the ethoxysilane. With respect to the linkers, their structural integrity and mobility are as easy to investigate by classical CP/MAS and high-resolution magic angle spinning (HRMAS) NMR techniques, as their adsorption behavior. Even electrostatic bonding to the support via phosphonium groups can be proven by solid-state NMR. For the immobilized catalysts, leaching, and even “horizontal” translational mobility effects, as probed by HRMAS NMR under “realistic conditions” in the presence of solvents, are described.     

New insights into Hansen's solubility parameters

Correlations among the three components, δ_d² (dispersion), δ_h² (hydrogen‐bonding), and δ_p² (polar) that make up the Hansen solubility parameter equation, δ_o² = δ_d² + δ_h² + δ_p², have been analyzed for a large number of organic solvents. A relationship is found that enables δ_h and δ_p to be estimated if δ_o and δ_d are known. This relationship is applied to a variety of common polymers and remarkably good agreement is obtained with tabulated values for δ_h and δ_p. Additional correlations are found that can be expressed in approximate functional form. The analysis also reveals relationships, expressed as inequalities, among the parameters that limit their range of possible values. © 2004 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 42: 4337–4343, 2004     

A theoretical insight into the photophysics of psoralen

Psoralen photophysics has been studied on quantum chemistry grounds using the multiconfigurational second-order perturbation method CASPT2. Absorption and emission spectra of the system have been rationalized by computing the energies and properties of the low-lying singlet and triplet excited states. The S1 pipi* state has been determined to be responsible of the lowest absorption and fluorescence bands and to initially carry the population in the photophysical processes related to the phototherapeutic properties of psoralen derivatives. The low-lying T1 pipi* state is, on the other hand, protagonist of the phosphorescence, and its prevalent role in the reactivity of psoralen is suggested to be related to the elongation of the pyrone ring C3-C4 bond, where the spin density is distributed on both carbon atoms. Analysis of energy gaps and spin-orbit coupling elements indicates that the efficient photophysical process leading to the population of the lowest triplet state does not take place at the Franck-Condon region but along the S1 relaxation path.     

New insights into flavivirus biology: the influence of pH over interactions between prM and E proteins

Diseases caused by flaviviruses, such as dengue and zika, are globally recognized as major threats. During infection, a critical point in their replicative cycle is the maturation step, which occurs throughout the cellular exocytic pathway. This step is a pH-dependent process that involves the modification of the viral envelope by converting prM (pre-membrane) into M (membrane) proteins with the release of a “pr peptide”. After this reaction, the pr peptides remain bound to the viral envelope while the virions cross the acidic trans-Golgi network, and are released only at neutral pH after secretion of the virus particles. Despite this current knowledge, the molecular basis of the flavivirus maturation step is largely unknown. Here, based on the crystal structure of the dengue pr–E complex (“pr peptide” bound to virus envelope protein) and using molecular dynamics simulations, we found that the pH shift from acidic to neutral yields considerable structural changes in the system. Dynamic cross correlation maps and root mean square deviation analyses revealed that the pr–E junction is clearly unstable under neutral pH. Secondary structure analysis also revealed that the fusion loop region, present in the E protein, is sensitive to pH and tends to unstructure at a neutral environment. Moreover, we found that five residues present in the E protein, Gly¹⁰², His²⁴⁴, Thr⁷⁰, Thr⁶⁸ and Asn⁶⁷ are critical to confer stability to the pr–E complex while inside the Golgi apparatus. This work brings details about the dynamical behavior of the pr–E system, helps to better understand the flavivirus biology and may also be of use in the development of novel antiviral strategies.     

High-throughput,multiparameter analysis of single cells

Heterogeneity of cell populations in various biological systems has been widely recognized, and the highly heterogeneous nature of cancer cells has been emerging with clinical relevance. Single-cell analysis using a combination of high-throughput and multiparameter approaches is capable of reflecting cell-to-cell variability, and at the same time of unraveling the complexity and interdependence of cellular processes in the individual cells of a heterogeneous population. In this review, analytical methods and microfluidic tools commonly used for high-throughput, multiparameter single-cell analysis of DNA, RNA, and proteins are discussed. Applications and limitations of currently available technologies for cancer research and diagnostics are reviewed in the light of the ultimate goal to establish clinically applicable assays.

New insights on hydrogen evolution at Au single crystal electrodes

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New insights into the mechanism of flow-electrode capacitive deionization

We report on Faradaic reactions producing H⁺ (anode) and OH^– (cathode) in flow-electrode capacitive deionization (FCDI) operated at 1.2 V. These reactions underline an additional electrodialytical desalination mechanism within capacitive deionization, which proceeds in parallel to the known electrosorption mechanism. Examination of flow-electrodes (100 ml each, 5% (wt) activated carbon) during FCDI (121 cm² effective membrane area) of 150 ml, 4 g/l NaCl solution revealed that significant amounts of Na⁺ and Cl⁻ ions (up to 50% and 30% of Cl⁻ and Na⁺, respectively) were not adsorbed in the activated carbon particles but were rather dissolved in the aqueous phase of the flow-electrodes. Production of acid (resulting in pH ≈ 1.5) and base (pH ≈ 12.5) in the flow-anode and -cathode solutions was observed during the operation. Reverse pH behaviors were obtained during the regeneration of the flow-electrodes by potential reversal. pH neutralization of the flow-electrode solutions resulted in a sharp increase in both the desalination rate and the electric current of the FCDI cell. Reacting NaOH and HCl in a short-circuited FCDI cell resulted in NaCl production in the water compartment and pH neutralization of both flow-electrodes.Apparently reversible Faradaic reactions that occur on the flow electrodes in the FCDI can be dependent on the properties of the carbon material, electrolyte composition and applied operational parameters (e.g. cell potential) and need to be studied in further detailed investigations.     

New insights into the conformational properties of α-C-glucosides

A series of alkyl α-d-C-glucopyranosides were synthesized and their conformational properties analyzed by CD and NMR spectroscopy. The conformational analysis revealed that the hydroxymethyl group populations (torsion angle ω, O1–C1–C2–O2) and those around the C-glucopyranosidic bond (torsion angle Φ; O2–C6–C7–C8) depend on the structural nature of the C-aglycon. The gt and the exo–syn populations increased as the C-aglycon became more substituted. Linear correlations between these rotational populations and proton chemical shifts versus the Taft’s steric parameters revealed the significant role of the C-aglycon in the overall conformation of C-glucosides. The stereoelectronic exo-deoxoanomeric effect, affecting the rotation of the hydroxymethyl group, becomes more important as the steric hindrance of motion increases. A pseudo-anomer rotational comparison study was also performed.