Towards Zwitterionic Oligonucleotides with Improved Properties: the NAA/LNA-Gapmer Approach

Oligonucleotides (ON) are promising therapeutic candidates, for instance by blocking endogenous mRNA (antisense mechanism). However, ON usually require structural modifications of the native nucleic acid backbone to ensure satisfying pharmacokinetic properties. One such strategy to design novel antisense oligonucleotides is to replace native phosphate diester units by positively charged artificial linkages, thus leading to (partially) zwitterionic backbone structures. Herein, we report a “gapmer” architecture comprised of one zwitterionic central segment (“gap”) containing nucleosyl amino acid (NAA) modifications and two outer segments of locked nucleic acid (LNA). This NAA/LNA-gapmer approach furnished a partially zwitterionic ON with optimised properties: i) the formation of stable ON-RNA duplexes with base-pairing fidelity and superior target selectivity at 37 °C; and ii) excellent stability in complex biological media. Overall, the NAA/LNA-gapmer approach is thus established as a strategy to design partially zwitterionic ON for the future development of novel antisense agents.     

The Quest for Xenobiotic Enzymes: From New Enzymes for Chemistry to a Novel Chemistry of Life

Enzyme engineering has made impressive progress in the past decades, paving the way for the widespread use of enzymes for various purposes. In contrast to “classical” enzyme engineering, which focuses on optimizing specific properties of natural enzymes, a more recent trend towards the creation of artificial enzymes that catalyze fundamentally distinct, new-to-nature reactions is observable. While approaches for creating such enzymes differ significantly, they share the common goal of enabling biocatalytic novelty to broaden the range of applications for enzymes. Although most artificial enzymes reported to date are only moderately active and barely function in vivo, they have the potential to endow cells with capabilities that were previously out of reach and thus herald a new wave of “functional xenobiology”. Herein, we highlight recent developments in the field of artificial enzymes with a particular focus on challenges and opportunities for their use in xenobiology.     

Synthesis of Lysozyme–Metallacarborane Conjugates and the Effect of Boron Cluster Modification on Protein Structure and Function

Two complementary methods, “in solution” and “in solid state”, for the synthesis of lysozyme modified with metallacarborane (cobalt bis(dicarbollide), Co(C₂B₉H₁₁)₂^2?) were developed. As metallacarborane donors, oxonium adducts of cobalt bis(dicarbollide) and 1,4‐dioxane or tetrahydropyran were used. The physicochemical and biochemical properties of the obtained lysozyme–metallacarborane conjugates were studied for changes in secondary and tertiary structure, aggregation behavior, and biological activity. Only minor changes in primary, secondary, and tertiary protein structure were observed, caused by the single substitution of metallacarborane on lysozyme. However, the modification produced significant changes in lysozyme enzymatic activity and a tendency toward time‐ and temperature‐dependent aggregation.     

Structural Insights into Conformation Differences between DNA/TNA and RNA/TNA Chimeric Duplexes

Threose nucleic acid (TNA) is an artificial genetic polymer capable of heredity and evolution, and is studied in the context of RNA chemical etiology. It has a four‐carbon threose backbone in place of the five‐carbon ribose of natural nucleic acids, yet forms stable antiparallel complementary Watson–Crick homoduplexes and heteroduplexes with DNA and RNA. TNA base‐pairs more favorably with RNA than with DNA but the reason is unknown. Here, we employed NMR, ITC, UV, and CD to probe the structural and dynamic properties of heteroduplexes of RNA/TNA and DNA/TNA. The results indicate that TNA templates the structure of heteroduplexes, thereby forcing an A‐like helical geometry. NMR measurement of kinetic and thermodynamic parameters for individual base pair opening events reveal unexpected asymmetric “breathing” fluctuations of the DNA/TNA helix. The results suggest that DNA is unable to fully adapt to the conformational constraints of the rigid TNA backbone and that nucleic acid breathing dynamics are determined from both backbone and base contributions.     

Flame retardant polyesters based on bromine derivatives

Unsaturated polyesters containing halogens in the backbone of the polymer chain were synthesized. Derivatives used had brominated functional groups such as tetrabromobisphenol A, tetrabromophthalic anhydride, and dibromoneopentyl glycol. These were compared to chlorine-containing polyesters based on HET acid and tetrachlorophthalic anhydride and an additive exhibiting a synergistic effect of phosphorus and halogen, trichloroethyl phosphate. Optical properties before and after artificial weathering (up to 1650 hr), were determined on cast specimens, and mechanical properties (flexural strength, modulus, and hardness) and flame retardancy were determined on glass fiber laminates. The flammability tests consisted of the “oxygen index” and the “self-extinguishing time” tests, which were correlated for various halogen concentrations and different compositions. The efficiency of bromine in flame retardancy has been found to be much higher than that of chlorine. The minimal concentration of bromine required for self-extinguishing is 10–12%, as compared with 20–25% for chlorine. A general performance index for polyesters has been derived which incorporates both flame and weather resistivity factors of the modified polyesters.     

Quantitative analysis of branching in poly(ortho ester) networks

A direct method to quantify the branching attributable to fully reacted hexane-1,2,6-triol (HT) in poly(ortho ester) networks is reported. The method was based on the presence of free hydroxyl groups on incompletely reacted HT in the poly(ortho ester) matrix that were “tagged” prior to matrix hydrolysis. HT molecules that were completely reacted, i.e., acting as cross-linker, within the polymer matrix would have no free hydroxyl groups available for “tagging” and posthydrolysis would be present as free HT. Experimentally, the matrix was swollen in p-dioxane, then phenyl isocyanate (PhCNO) was added to “tag” the hydroxyl groups in the presence of a stannous octoate catalyst. After removal of excess PhCNO, the matrix was hydrolyzed under mild acidic conditions. Any “untagged” HT was subsequently derivatized into trimethylsilyl ethers and analyzed by gas chromatography (GC). The level of branching determined by this direct chemical method correlated well with conventional swell ratio measurements. Furthermore, the extent of hydrolysis of the polymer backbone covalent bonds were also measured by this method since the resulting hydroxyl groups were “tagged” by PhCNO. All polyols were analyzed simultaneously by GC. Application of this method in characterization of the solid-state hydrolysis of a poly(ortho ester) network was demonstrated. The degradation profiles of the branch sites and other backbone ortho ester bonds showed that the hydrolysis was initially slow, becoming progressively rapid. © 1993 John Wiley & Sons, Inc.     

Artificial Maturation of the Highly Active Heterodimeric [FeFe] Hydrogenase from Desulfovibrio desulfuricans ATCC 7757

Hydrogenases catalyze the reduction of protons and oxidation of molecular hydrogen with high turnover frequencies and low overpotentials under ambient conditions. The heterodimeric [FeFe] hydrogenase from Desulfovibrio desulfuricans has an exceptionally high activity, and can be purified aerobically in an oxygen-stable inactive state. Recently, it was demonstrated that monomeric [FeFe] hydrogenases produced recombinantly in Escherichia coli can be artificially maturated by simply incubating the inactive “apo” enzymes with the synthetic [2Fe] cofactor mimic [Fe₂(adt)(CO)₄(CN)₂]²⁻. Here, we use the same technique to produce the heterodimeric “apo” hydrogenase from D. desulfuricans in E. coli with a high yield and purity, and maturate the “apo” enzyme with [Fe₂(adt)(CO)₄(CN)₂]²⁻ to generate fully active “holo” enzyme. Interestingly, the rate of the artificial maturation process with D. desulfuricans is significantly slower than that for all other hydrogenases tested so far. The artificially maturated enzyme is spectroscopically and electrochemically identical to the native enzyme and shows high rates of hydrogen production (3700 s⁻¹) and hydrogen oxidation (63,000 s⁻¹). We expect that our highly efficient production method will facilitate future studies of this enzyme and other related [FeFe] hydrogenases from Desulfovibrio species.     

New poly(acrylic acid) containing segmented copolymer structures by combination of “click” chemistry and atom transfer radical polymerization

In this paper, the combination of atom transfer radical polymerization (ATRP) of 1-ethoxyethyl acrylate (EEA) and the copper(I) catalyzed “click” 1,3-dipolar cycloaddition reaction of azides and terminal alkynes was evaluated as a method to synthesize diverse amphiphilic copolymer structures. Using the 1-ethoxyethyl protecting group strategy, the application field was broadened with the synthesis of complex polymer structures containing poly(acrylic acid) (PAA) segments. A modular approach has been used: polymers with alkyne functionalities as well as azide functionalities have been synthesized. These polymers were subsequently “clicked” together to yield block copolymers. Furthermore, graft copolymers were synthesized by grafting alkyne-containing polymers onto a polymer backbone with multiple azide functions using the combination of ATRP and “click” reactions.     

Bioelectrochemistry and the Singularity Point “I Robot”?**

In his book “The Singularity Is Near: When Humans Transcend Biology” Ray Kurtzweil described the concept of artificial intelligence in an unorthodox way. It describes how humans will interface with machines and eventually will become one with machines. Hence, we can define a time point, which is a theoretical point when humans and machines will become one. In order to reach this point, a successful interface between the biological/organic to the inorganic matter is needed. In order for these pieces of “machinery” to communicate between one another, an exchange of matter and energy should be achieved. Electron transfer between the inorganic and organic has been the mission of bioelectrochemical systems in the past few decades. Enzymes, antibodies and nucleic acids attachment to electrodes has been achieved more than three decades ago. In the following assay I am reviewing the different advances that were made towards an integration between biological entities to inorganic ones.     

Iterative Nonproteinogenic Residue Incorporation Yields α/β‐Peptides with a Helix–Loop–Helix Tertiary Structure and High Affinity for VEGF

Inhibition of specific protein–protein interactions is attractive for a range of therapeutic applications, but the large and irregularly shaped contact surfaces involved in many such interactions make it challenging to design synthetic antagonists. Here, we describe the development of backbone‐modified peptides containing both α‐ and β‐amino acid residues (α/β‐peptides) that target the receptor‐binding surface of vascular endothelial growth factor (VEGF). Our approach is based on the Z‐domain, which adopts a three‐helix bundle tertiary structure. We show how a two‐helix “mini‐Z‐domain” can be modified to contain β and other nonproteinogenic residues while retaining the target‐binding epitope by using iterative unnatural residue incorporation. The resulting α/β‐peptides are less susceptible to proteolysis than is their parent α‐peptide, and some of these α/β‐peptides match the full‐length Z‐domain in terms of affinity for receptor‐recognition surfaces on the VEGF homodimer.     

Sampled-data feedback control of a binary distillation column

The design of sampled-data controllers for a binary distillation column was studied for several types of controllers and for various control tray locations, sampling rates and disturbances. Minimal prototype control was superior to continuous PI control under some conditions but, as expected, was only satisfactory for a specified disturbance. dual “A” control scheme that compensates for both setpoint and load disturbances was developed. Two separate algorithms are implemented simultaneously, one receiving its input signal from the setpoint and the other from the measured variable. Dual control is superior to continuous control, particularly when both disturbances are encountered at the same time, and is superior to the “switching” method proposed by Mosler et al.     

Miscibility,morphology, structure,and properties of porous cellulose–soy protein isolate hybrid hydrogels

Porous hybrid hydrogels were fabricated by mixing cellulose (CEL) and soy protein isolate (SPI) solutions, followed by crosslinking with epichlorohydrin. Their miscibility, morphology, structure, and properties were investigated by wide‐angle X‐ray diffraction, thermogravimetric analysis, scanning electron microscopy, dynamic mechanical analysis, rheological measurement, and swelling tests. The results show that CEL performed as a “scaffold” of pore walls and contributed to the good mechanical properties, while SPI performed the role of an “extender” of pore size and was responsible for the high water absorbency. The incorporation of CEL (stiff chains) and SPI (hydrophilic groups) in the hybrid hydrogel constructed the porous structure. This work provides a method for the fabrication of hydrogels with porous structure through the combination of a stiff material as a “scaffold” and a hydrophilic material as an “extender.” © 2016 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2016 , 133, 43853.     

A PEG-based oligomer as a backbone replacement for surface-exposed loops in a protein tertiary structure

PEGged out: Poly(ethylene glycol), a simple biocompatible polymer, can replace natural loop segments in a 56-residue protein domain with a well-defined tertiary structure. Biophysical characterization of chimeras of the protein GB1 coupled with molecular dynamics simulations show that PEG enhances local backbone torsional freedom without compromising the overall protein fold or function.     

Ferrocenylanthracene polymers: the direct synthesis using “Lawesson’s Reagent”

A method for polymerizing ferrocenylanthraquinone compounds was investigated, using a methodology developed originally for the polymerization of 9,10-anthracenedithiols. This procedure uses an in situ reaction of “Lawesson’s Reagent” (p-methoxyphenylthionophosphine sulphide dimer), with anthraquinone allowing polymerization to proceed across the 9- and 10-positions of the ferrocenylanthraquinones resulting in polymers with pendant ferrocene groups on the polymer backbone. Monomers used for these reactions were anthraquinone (literature comparison), 2-ferrocenylanthraquinone and 2,6-diferrocenylanthraquinone.     

REVIEW OF VOLATILE ORGANIC COMPOUNDS DERIVED FROM POLYETHYLENE

The formation of volatile organic compounds (VOCs) in polyethylene (PE) is a topic of concern to industries involved in the packaging of items such as foodstuffs and pharmaceuticals that are sensitive to organoleptic contamination. This article reviews the available literature on VOCs that originate from PE during its manufacture, processing, storage, and service life. The package–product interactions that may occur between PE and packaged foodstuffs are also considered together with the wide range of methods for the analysis of VOCs. The following analytical methods are discussed: (i) sensory evaluation, (ii) chromatographic techniques and their associated sampling techniques, including the “hot-jar” method and dynamic headspace sampling, (iii) gas chromatography–olfactory sensing, and (iv) artificial olfaction or “electronic nose” technology.     

The thermal analyses of polymers. II. Thermomechanical analyses of segmented polyurethane elastomers

The development of new and more sensitive techniques in thermal analyses aids in a more complete understanding of the contributions of individual components in urethane elastomers regarding their mechanical and thermodynamic behavior. The behavior of various segments of the elastomers reported in this work illustrates a clearer interpretation of reasons for changes in mechanical behavior caused by changes in heat capacity, volume and tensile properties; the gross changes previously reported for polyurethane properties as a function of temperature are also confirmed with a more exact definition of their origin. The sub-ambient temperature behavior and response of physical measurements near the melting point of the backbone polyol are largely a function of the so-called “soft block.” The soft block does not contribute to the mechanical properties above the melting point of the polyol unless some urethane segments from the diisocyanate and extender are structured into the soft block, that is, excess diisocyanate and extender are added to build in the “hard” block. The extender and isocyanate influence for both low and high temperature properties is observed by the lack of molecular fit imparted to the backbone polyol as well as some crystallinity in the polymer hard block. The usual T_g transition reported in urethane elastomers corresponds to a first-order transition in the polyester or polyether backbone.     

A Multi-Objective Approach for Protein Structure Prediction Based on an Energy Model and Backbone Angle Preferences

Protein structure prediction (PSP) is concerned with the prediction of protein tertiary structure from primary structure and is a challenging calculation problem. After decades of research effort, numerous solutions have been proposed for optimisation methods based on energy models. However, further investigation and improvement is still needed to increase the accuracy and similarity of structures. This study presents a novel backbone angle preference factor, which is one of the factors inducing protein folding. The proposed multiobjective optimisation approach simultaneously considers energy models and backbone angle preferences to solve the ab initio PSP. To prove the effectiveness of the multiobjective optimisation approach based on the energy models and backbone angle preferences, 75 amino acid sequences with lengths ranging from 22 to 88 amino acids were selected from the CB513 data set to be the benchmarks. The data sets were highly dissimilar, therefore indicating that they are meaningful. The experimental results showed that the root-mean-square deviation (RMSD) of the multiobjective optimization approach based on energy model and backbone angle preferences was superior to those of typical energy models, indicating that the proposed approach can facilitate the ab initio PSP.     

Mesoscopic coarse‐grained simulations of hydrophobic charge induction chromatography (HCIC) for protein purification

Mesoscopic coarse‐grained simulations are adopted to investigate interfacial mechanisms of hydrophobic charged induction chromatography for protein purification by regulating pH. Simulations results indicate that: (i) lysozyme can be adsorbed mainly with “top end‐on” and “bottom end‐on” orientation on hydrophobic surfaces, dominated by the two hydrophobic regions located at both ends of lysozyme's long axis. Elution from the “top end‐on” orientation is more difficult than that from the “bottom end‐on” orientation; (ii) a higher ligand density can get a faster adsorption rate and stronger adsorption. Interestingly, the effect of ligand density on the desorption is mainly determined by the distribution probability of the positively charged groups of ligands; (iii) a higher ionic strength can lead to a wider orientation distribution, a stronger adsorption and a lower elution rate. This work might provide an efficient way to optimize the operating conditions and designing novel ligands. © 2015 American Institute of Chemical Engineers AIChE J, 61: 2035–2047, 2015     

PAF receptor structure: A hypothesis

Different hypotheses of the structure of platelet-activating factor (PAF) receptor based on structure-activity relationships of agonists and antagonists are reviewed. For an agonistic effect, strong hydrophobic interactions and an ether function are required in position-1 of the glycerol backbone; chain length limitations and steric hindrance demand a small group in position-2. The unusual structural properties of non-PAF-like antagonists required 3-D electrostatic potential calculations. This method applied to seven potent antagonists suggests a strong “Cache-oreilles” (ear-muff) effect,i.e., two strong electronegative wells (isocontour at ?10 Kcal/mole) are located at 180° to each other and at a relatively constant distance. Initial consideration of the “Cache-oreilles” effect implied the structure of a bipolarized cylinder of 10–12 Å diameter for the receptor. However, very recent results on studies with agonists and antagonists structurally similar to PAF suggest that the receptor may in fact be a multi-polarized cylinder.     

A Universal Chemical Method for Rational Design of Protein-Based Nanoreactors**

Self-assembly of a monomeric protease to form a multi-subunit protein complex “proteasome” enables targeted protein degradation in living cells. Naturally occurring proteasomes serve as an inspiration and blueprint for the design of artificial protein-based nanoreactors. Here we disclose a general chemical strategy for the design of proteasome-like nanoreactors. Micelle-assisted protein labeling (MAPLab) technology along with the N-terminal bioconjugation strategy is utilized for the synthesis of a well-defined monodisperse self-assembling semi-synthetic protease. The designed protein is programmed to self-assemble into a proteasome-like nanostructure which preserves the functional properties of native protease.