Biocompatible Size‐Defined Dendrimer–Albumin Binding Protein Hybrid Materials as a Versatile Platform for Biomedical Applications

For the design of a biohybrid structure as a ligand‐tailored drug delivery system (DDS), it is highly sophisticated to fabricate a DDS based on smoothly controllable conjugation steps. This article reports on the synthesis and the characterization of biohybrid conjugates based on noncovalent conjugation between a multivalent biotinylated and PEGylated poly(amido amine) (PAMAM) dendrimer and a tetrameric streptavidin‐small protein binding scaffold. This protein binding scaffold (SA‐ABDwt) possesses nM affinity toward human serum albumin (HSA). Thus, well‐defined biohybrid structures, finalized by binding of one or two HSA molecules, are available at each conjugation step in a controlled molar ratio. Overall, these biohybrid assemblies can be used for (i) a controlled modification of dendrimers with the HSA molecules to increase their blood‐circulation half‐life and passive accumulation in tumor; (ii) rendering dendrimers a specific affinity to various ligands based on mutated ABD domain, thus replacing tedious dendrimer–antibody covalent coupling and purification procedures.

     

Interpretation of synchrotron radiation circular dichroism spectra of anionic, cationic, and zwitterionic dialanine forms

Electronic absorption and synchrotron radiation circular dichroism (SRCD) spectra of the anionic, cationic, and zwitterionic forms of L-alanyl-L-alanine (AA) in aqueous solutions were measured and interpreted by molecular dynamics (MD) and ab initio computations. Time-dependent density functional theory (TD DFT) was applied to predict the electronic excited states. The modeling enabled the assessment of the role of molecular conformation, charge, and interaction with the polar environment in the formation of the spectral shapes. Particularly, inclusion of explicit solvent molecules in the computations appeared to be imperative because of the participation of water orbitals in the amide electronic structure. Implicit dielectric continuum solvent models gave inferior results for clusters, especially at low-energy transitions. Because of the dispersion of transition energies, tens of water/AA clusters had to be averaged in order to obtain reasonable spectral shapes with a more realistic inhomogeneous broadening. The modeling explained most of the observed differences, as the anionic and zwitterionic SRCD spectra were similar and significantly different from the cationic spectrum. The greatest deviation between the experimental and theoretical curves observed for the lowest-energy negative anion signal can be explained by the limited precision of the TD DFT method, but also by the complex dynamics of the amine group. The results also indicate that differences in the experimental spectral shapes do not directly correlate with the peptide main-chain conformation. Future peptide and protein conformational studies based on circular dichroic spectroscopy can be reliable only if such effects of molecular dynamics, solvent structure, and polar solvent-solute interactions are taken into account.     

Hydroaminomethylation with novel rhodium-carbene complexes: an efficient catalytic approach to pharmaceuticals

Starting from [{Rh(cod)Cl}(2)] and 1,3-dimesitylimidazole-2-ylidenes the novel [RhCl(cod)(carbene)] complexes 1-5 have been synthesized, characterized, and tested in the hydroaminomethylation of aromatic olefins. The influence of different ligands and reaction parameters on the catalytic activity was investigated in detail applying 1,1-diphenylethylene and piperidine as a model system. The scope and limitations of the novel catalysts is shown in the preparation of 16 biologically active 1-amino-3,3-diarylpropenes. In general, high chemo- and regioselectivity as well as good yields of the desired products were achieved.     

Automation of solid-phase microextraction on a 96-well plate format

Studies have been performed assessing the feasibility and characterizing the automation of solid-phase microextraction (SPME) on a multi-well plate format. Four polycyclic aromatic hydrocarbons (PAHs), naphthalene, fluorene, anthracene and fluoranthene, were chosen as test analytes to demonstrate the technique due to their favorable partition coefficients, K(fw), between polydimethylsiloxane (PDMS) extraction phases and water. Four different PDMS configurations were investigated regarding their suitability. These included (i) a PDMS membrane; (ii) a multi-fiber device containing lengths of PDMS-coated flexible wire; (iii) a stainless steel pin covered with silicone hollow fiber membrane and (iv) commercial PDMS-coated flexible metal fiber assemblies. Of these configurations, the stainless steel pin covered with silicone tubing was chosen as a robust alternative. An array of 96 SPME devices that can be placed simultaneously into a 96-well plate was constructed to demonstrate the high-throughput potential when performing multiple microextractions in parallel. Different agitation methods were assessed including magnetic stirring, sonication, and orbital shaking at different speeds. Orbital shaking whilst holding the SPME device in a stationary position provided the optimum agitation conditions for liquid SPME. Once the analytes had been extracted, desorption of the analytes into an appropriate solvent was investigated. Liquid-phase SPME and solvent desorption on the multi-well plate format is shown to be a viable alternative for automated high-throughput SPME analysis compatible with both gas- and liquid-chromatography platforms.     

Semisynthetic Derivatives of Selected Amaryllidaceae Alkaloids as a New Class of Antimycobacterial Agents

The search for novel antimycobacterial drugs is a matter of urgency, since tuberculosis is still one of the top ten causes of death from a single infectious agent, killing more than 1.4 million people worldwide each year. Nine Amaryllidaceae alkaloids (AAs) of various structural types have been screened for their antimycobacterial activity. Unfortunately, all were considered inactive, and thus a pilot series of aromatic esters of galanthamine, 3-O-methylpancracine, vittatine and maritidine were synthesized to increase biological activity. The semisynthetic derivatives of AAs were screened for their in vitro antimycobacterial activity against Mycobacterium tuberculosis H37Ra and two other mycobacterial strains (M. aurum, M. smegmatis) using a modified Microplate Alamar Blue Assay. The most active compounds were also studied for their in vitro hepatotoxicity on the hepatocellular carcinoma cell line HepG2. In general, the derivatization of the original AAs was associated with a significant increase in antimycobacterial activity. Several pilot derivatives were identified as compounds with micromolar MICs against M. tuberculosis H37Ra. Two derivatives of galanthamine, 1i and 1r, were selected for further structure optimalization to increase the selectivity index.     

Interaction of Love waves with coupled cavity modes in a 2D holey phononic crystal

The interaction of Love waves with square array of pillars deposited on a cavity defined in a 2D holey phononic crystal is numerically investigated using Finite Element Method. First, the existence of SH surface modes is demonstrated separately for phononic crystals that consist of square arrayed holes, or rectangular arrayed Ni pillars, respectively in, or on, a SiO₂ film deposited on a ST-cut quartz substrate. The coupling between SH modes and torsional mode in pillars induces a transmission dip that occurs at a frequency located in the range of the band-gap of the holey phononic crystal. Second, a cavity is constructed by removing lines of holes in the holey phononic crystal and results in a transmission peak that matches the dip. The optimal geometrical parameters enable us to create a coupling of the cavity mode and the localized pillar mode by introducing lines of pillars into the cavity, which significantly improved the efficiency of the cavity without increasing the crystal size. The obtained results will pave the way to implement advanced designs of high-performance electroacoustic sensors based on coupling modes in phononic crystals.     

Modal resonant ultrasound spectroscopy for ferroelastics

Recent experimental and theoretical improvements of resonant ultrasound spectroscopy (RUS) are summarized to investigate elastic constants of phases in shape memory alloys. The proposed inversion procedure, described in this work, is particularly suitable to reliable evaluation of the temperature dependence of elastic constants of low-symmetry ferroelastic materials which may be strongly elastically anisotropic and tend to exist in twinned forms. The method is applicable even for the evaluation of single-crystal elastic constants from RUS measurements on microtwinned crystals, since it involves a homogenization algorithm based on the macroscopic deformation response of the layered structure. This potentially allows performing meaningful acoustic studies on samples with a general submicron-size layered structure.     

Engineering Escherichia coli for Fermentative Dihydrogen Production: Potential Role of NADH-Ferredoxin Oxidoreductase from the Hydrogenosome of Anaerobic Protozoa

Trichomonas vaginalis generates reduced ferredoxin within a unique subcellular organelle, hydrogenosome that is used as a reductant for H₂ production. Pyruvate ferredoxin oxidoreductase and NADH dehydrogenase (NADH-DH) are the two enzymes catalyzing the production of reduced ferredoxin. The genes encoding the two subunits of NADH-DH were cloned and expressed in Escherichia coli. Kinetic properties of the recombinant heterodimer were similar to that of the native enzyme from the hydrogenosome. The recombinant holoenzyme contained 2.15 non-heme iron and 1.95 acid-labile sulfur atoms per heterodimer. The EPR spectrum of the dithionite-reduced protein revealed a [2Fe–2S] cluster with a rhombic symmetry of g _xyz?=?1.917, 1.951, and 2.009 corresponding to cluster N1a of the respiratory complex I. Based on the Fe content, absorption spectrum, and the EPR spectrum of the purified small subunit, the [2Fe–2S] cluster was located in the small subunit of the holoenzyme. This recombinant NADH-DH oxidized NADH and reduced low redox potential electron carriers, such as viologen dyes as well as Clostridium ferredoxin that can couple to hydrogenase for H₂ production from NADH. These results show that this unique hydrogenosome NADH dehydrogenase with a critical role in H₂ evolution in the hydrogenosome can be produced with near-native properties in E. coli for metabolic engineering of the bacterium towards developing a dark fermentation process for conversion of biomass-derived sugars to H₂ as an energy source.     

Hydrophilic interaction liquid chromatography – charged aerosol detection as a straightforward solution for simultaneous analysis of ascorbic acid and dehydroascorbic acid

Ascorbic acid (AA) and dehydroascorbic acid (DHA) are small polar molecules difficult to be retained in conventional chromatographic RP systems. Hydrophilic interaction liquid chromatography (HILIC) using Obelisk R (100 × 3.2 mm, 5 μm, Sielc) analytical column and isocratic elution by ammonium acetate buffer pH 4.2 was found to be successful at this task, while other tested HILIC columns – Obelisk N (100 × 3.2 mm, 5 μm, Sielc) and Luna HILIC (100 × 3.0 mm, 3 μm, Phenomenex) were unsuccessful for the purposes of analysis. Charged aerosol detection (CAD) has recently become a new alternative universal detection system in HPLC, and was extremely convenient for the simultaneous analysis of AA and DHA without the need of subtraction approach and oxidation/reduction step. CAD response was found linear in defined range in spite of the fact that CAD is designated as non-linear detection method. A simple and fast HILIC-CAD method was applied for the analysis of pharmaceutical preparations containing AA. Method validation was performed including parameters of precision, accuracy, linearity, limit of detection and limit of quantitation (LOQ). The method was fast, accurate and precise for both detectors with LOQ_AA 5 μg/ml for UV detection and 10 μg/ml for CAD, respectively. DHA was detected only by CAD within tested concentration range with LOQ_DHA 1 μg/ml.     

Reaction rate theory for supramolecular kinetics: application to protein aggregation

ABSTRACT

Probing reaction mechanisms of supramolecular processes in soft and biological matter, such as protein aggregation, is inherently challenging. This is because these processes involve multiple molecular mechanisms that are associated with the rearrangement of large numbers of weak bonds, resulting in complex free energy landscapes with many kinetic barriers. Reaction rate measurements at different temperatures can offer unprecedented insights into the underlying molecular mechanisms. However, to be able to interpret such measurements, a key challenge is to establish which properties of the complex free energy landscapes are probed by the reaction rate. Here, we present a reaction rate theory for supramolecular kinetics based on Kramers theory of diffusive reactions over multiple kinetic barriers. We find that reaction rates for protein aggregation are of the Arrhenius–Eyring type and that the associated activation energies probe only one relevant barrier along the respective free energy landscapes. We apply this advancement to interpret, in experiments and in coarse-grained computer simulations, reaction rates of amyloid aggregation in terms of molecular mechanisms and associated thermodynamic signatures. These results suggest a practical extension of the concept of rate-determining steps for complex supramolecular processes and establish a general platform for probing the underlying energy landscape using kinetic measurements.