Elucidating Diphosphoinositol Polyphosphate Function with Nonhydrolyzable Analogues

The diphosphoinositol polyphosphates (PP‐IPs) represent a novel class of high‐energy phosphate‐containing messengers which control a wide variety of cellular processes. It is thought that PP‐IPs exert their pleiotropic effects as allosteric regulators and through pyrophosphorylation of protein substrates. However, most details of PP‐IP signaling have remained elusive because of a paucity of suitable tools. We describe the synthesis of PP‐IP bisphosphonate analogues (PCP‐IPs), which are resistant to chemical and biochemical degradation. While the two regioisomers 1PCP‐IP₅ and 5PCP‐IP₅ inhibited Akt phosphorylation with similar potencies, 1PCP‐IP₅ was much more effective at inhibiting its cognate phosphatase hDIPP1. Furthermore, the PCP analogues inhibit protein pyrophosphorylation because of their inability to transfer the β‐phosphoryl group, and thus enable the distinction between PP‐IP signaling mechanisms. As such, the PCP analogues will find widespread applications for the structural and biochemical characterization of PP‐IP signaling properties.     

Synthesis of Highly Selective Submicromolar Microcystin‐Based Inhibitors of Protein Phosphatase (PP)2A over PP1

Research and therapeutic targeting of the phosphoserine/threonine phosphatases PP1 and PP2A is hindered by the lack of selective inhibitors. The microcystin (MC) natural toxins target both phosphatases with equal potency, and their complex synthesis has complicated structure–activity relationship studies in the past. We report herein the synthesis and biochemical evaluation of 11 MC analogues, which was accomplished through an efficient strategy combining solid‐ and solution‐phase approaches. Our approach led to the first MC analogue with submicromolar inhibitory potency that is strongly selective for PP2A over PP1 and does not require the complex lipophilic Adda group. Through mutational and structural analyses, we identified a new key element for binding, as well as reasons for the selectivity. This work gives unprecedented insight into how selectivity between these phosphatases can be achieved with MC analogues.     

The Bisphosphonate Odyssey. A Journey from Chemistry to the Clinic

Bisphosphonates are chemically stable analogues of inorganic pyrophosphate, which are resistant to breakdown by enzymatic hydrolysis. Bisphosphonates bind to bone mineral and inhibit the resorption of living bone. The biological effects of bisphosphonates on calcium metabolism were originally ascribed to their physico-chemical effects to impede the dissolution of hydroxyapatite crystals. Although such effects may contribute to their overall action, their effects on cells are probably of greater importance, particularly for the more potent compounds. The marked structure-activity relationships observed among more complex compounds indicate that the pharmacophore required for maximal activity depends not only upon the bisphosphonate moiety but also on key additional features, especially nitrogen substitution in alkyl or heterocyclic side chains.     

Dissecting the activation of insulin degrading enzyme by inositol pyrophosphates and their bisphosphonate analogs

Inositol poly- and pyrophosphates (InsPs and PP-InsPs) are densely phosphorylated eukaryotic messengers, which are involved in numerous cellular processes. To elucidate their signaling functions at the molecular level, non-hydrolyzable bisphosphonate analogs of inositol pyrophosphates, PCP-InsPs, have been instrumental. Here, an efficient synthetic strategy to obtain these analogs in unprecedented quantities is described – relying on the use of combined phosphate ester-phosphoramidite reagents. The PCP-analogs, alongside their natural counterparts, were applied to investigate their regulatory effect on insulin-degrading enzyme (IDE), using a range of biochemical, biophysical and computational methods. A unique interplay between IDE, its substrates and the PP-InsPs was uncovered, in which the PP-InsPs differentially modulated the activity of the enzyme towards short peptide substrates. Aided by molecular docking and molecular dynamics simulations, a flexible binding mode for the InsPs/PP-InsPs was identified at the anion binding site of IDE. Targeting IDE for therapeutic purposes should thus take regulation by endogenous PP-InsP metabolites into account.

An efficient synthesis of non-hydrolyzable inositol pyrophosphate analogs was developed to subsequently investigate the regulation of insulin-degrading enzyme by these hyperphosphorylated signaling molecules.     

Simultaneous determination of inositol and inositol phosphates in complex biological matrices: quantitative ion‐exchange chromatography/tandem mass spectrometry

myo‐Inositol (Ins) and myo‐inositol phosphates (InsPs) are widely distributed in plants and animals. The evaluation of the distribution of Ins and InsPs in cells and plant sources can impact the understanding of their role in nutrition, cellular processes and diseases, and how they may be modulated by diet. We developed an anion‐exchange chromatography/tandem mass spectrometry (HPLC/ESI‐MS/MS) method for the separation and simultaneous quantitation of Ins and different naturally occurring phosphorylated inositol compounds. Chromatographic separation was achieved in 30 min on a commercial anion‐exchange column (0.5 × 150 mm) using a gradient of 200 mM ammonium carbonate buffer (pH 9.0) and 5% methanol in H₂O. Analytes were identified by selective reaction monitoring using a triple quadrupole mass spectrometer in negative ion electrospray ionization mode. Adenosine 5′‐monophosphate was used as a general internal standard for quantitation. Detection is linear in the range of 0.25–400 pmol for Ins, InsP₁, InsP₄, and InsP₅, 40–400 pmol for InsP₂ and InsP₃, and 60–400 pmol for InsP₆, with a minimum r² > 0.994. The limit of detection is 0.25 pmol with a signal‐to‐noise ratio of 10:1 for all analytes. The intra‐day and inter‐day variations were within 17% at three concentration levels. Recovery values for the seven analytes spiked into extraction solution or different matrices were between 63 and 121%. Using this approach, Ins and InsPs were measured in three different plant samples and in cultured cells, illustrating significant differences in the distribution of inositol compounds in food samples compared to cells and between cell types. Copyright © 2009 John Wiley & Sons, Ltd.     

A Biocatalytic Cascade for Versatile One‐Pot Modification of mRNA Starting from Methionine Analogues

Methyltransferases have proven useful to install functional groups site‐specifically in different classes of biomolecules when analogues of their cosubstrate S‐adenosyl‐l ‐methionine (AdoMet) are available. Methyltransferases have been used to address different classes of RNA molecules selectively and site‐specifically, which is indispensable for biophysical and mechanistic studies as well as labeling in the complex cellular environment. However, the AdoMet analogues are not cell‐permeable, thus preventing implementation of this strategy in cells. We present a two‐step enzymatic cascade for site‐specific mRNA modification starting from stable methionine analogues. Our approach combines the enzymatic synthesis of AdoMet with modification of the 5′ cap by a specific RNA methyltransferase in one pot. We demonstrate that a substrate panel including alkene, alkyne, and azido functionalities can be used and further derivatized in different types of click reactions.     

Enhanced rigidity of recycled polypropylene from packaging waste by compounding with talc and high‐ crystallinity polypropylene

Polypropylene (PP) constituted 30% of the collected material in a Swedish collection system for rigid plastic packaging waste. The PP fraction was however a complex mixture of grades with widely different properties. In order to enhance the rigidity of the recycled PP, modified grades were prepared by compounding with talc and/or a virgin high‐crystallinity PP grade. Adding 20–40% of high‐crystallinity PP enhanced the stiffness and yield strength without impairing the impact resistance. A composite material consisting of 20% of this grade, 20% talc and 60% recycled PP gave mechanical properties similar to those of a commercial talc‐filled PP compound used for demanding engineering applications. The present study demonstrates that recycled PP derived from post‐consumer packaging waste can also be made useful for demanding engineering applications. Copyright © 2001 John Wiley & Sons, Ltd.     

Chelation of vanadium(V) by difluoromethylene bisphosphonate, a structural analogue of pyrophosphate

The structural and functional analogy between difluoromethylene bisphosphonate (CF2PP) and pyrophosphate (PPi) is investigated in a reaction with V(V) in the form of vanadate. The reaction of CF2PP with vanadate was investigated using 1.00 M KCl as supporting electrolyte over the ranges 3 < or = [CF2PP] < or = 60 mM and 2.06 < or = pH < or = 11.80. 51V, 19F, and 31P NMR spectroscopic studies showed that a 1:1 species was formed with an H+-dependent formation constant of 110 M-1 at pH 7.22. Results of solution experiments and ab initio calculations are consistent with CF2PP coordinating V(V) in a bidentate manner, as previously reported for PPi. Below pH 4, a minor complex forms, which is consistent with a 1:2 stoichiometry. This complex was also observed with pyrophosphate. The X-ray crystal structure of the monoprotonated difluoromethylene bisphosphonate anion (H[CF2PP]3-)-toludine complex is presented. The H[CF2PP]3- anion crystallized in the triclinic space group P with a = 12.7629(7) A, b = 13.3992(7) A, c = 17.1002(9) A, and V = 2584.4(2) A3, and Z = 2. Sheets of the layers of anions are connected through a network of H-bonds and separated by a layer of toludine cations. The structural features are investigated, and the CF2PP anion was found to be longer and wider than the corresponding PPi. Given the larger size of this anion compared to PPi, the chelation affinity upon CF2 substitution was found to be 4-5-fold reduced at neutral pH.     

Morphology and properties of polypropylene/ethylene–octene copolymer/clay nanocomposites with double compatibilizers

The influence of nanoclay on the morphology and properties of the polypropylene (PP)/ethylene–octene block copolymer (EOC) blend with double compatibilizers of maleated PP (PP‐g‐MA) and maleated EOC (EOC‐g‐MA) was investigated and compared with the nanocomposites containing either PP‐g‐MA or EOC‐g‐MA as a compatibilizer. X‐ray diffraction, transmission electron microscopy, and scanning electron microscopy were utilized for morphological characterization in conjunction with dynamic mechanical thermal analysis, mechanical testing, and rheological evaluation of these nanocomposites. The results suggested that in the nanocomposite including both compatibilizers of PP‐g‐MA and EOC‐g‐MA, clay was dispersed as a mixed structure of intercalation and exfoliation in both phases of the polymer blend. Comparing the mechanical properties of the studied nanocomposite with nanocomposites of PP/EOC/PP‐g‐MA/clay and PP/EOC/EOC‐g‐MA/clay also indicated that the nanocomposite containing mixed compatibilizers displayed higher tensile modulus, tensile strength, and complex viscosity because of the better dispersion of clay in both phases. The results also confirmed the increased structural stability and reduced dispersed phase size of PP/EOC/PP‐g‐MA/EOC‐g‐MA blend in the presence of clay that proposed the compatibilization role of clay in this nanocomposite. Copyright © 2014 John Wiley & Sons, Ltd.     

Spherical Nucleic Acid Nanoparticle Conjugates Enhance G‐Quadruplex Formation and Increase Serum Protein Interactions

To understand the effect of three‐dimensional oligonucleotide structure on protein corona formation, we studied the identity and quantity of human serum proteins that bind to spherical nucleic acid (SNA) nanoparticle conjugates. SNAs exhibit cellular uptake properties that are remarkably different from those of linear nucleic acids, which have been related to their interaction with certain classes of proteins. Through a proteomic analysis, this work shows that the protein binding properties of SNAs are sequence‐specific and supports the conclusion that the oligonucleotide tertiary structure can significantly alter the chemical composition of the SNA protein corona. This knowledge will impact our understanding of how nucleic acid‐based nanostructures, and SNAs in particular, function in complex biological milieu.     

Morphology and mechanical behavior of isotactic polypropylene with different stereo‐defect distribution in injection molding

Large amount of work has been published on the isotacticity–properties relationship of isotactic polypropylene (iPP). However, the stereo‐defect distribution dependence of morphology and mechanical properties of iPP injection molding samples is still not clear. In this study, two different isotactic polypropylene (iPP) resins (PP‐A and PP‐B) with similar average isotacticity but different stereo‐defect distribution were selected to investigate the morphology evolution and mechanical properties (tensile and notching) of their injection molding samples using differential scanning calorimetry (DSC), dynamic mechanical analysis (DMA), 2D wide angle X‐ray diffraction (2D‐WAXD), and scanning electron microscope (SEM). The results of DMA showed that the molecular movement ability of PP‐A (with less uniform distribution of stereo‐defect) was stronger than that of PP‐B, meanwhile the analysis of DSC and SEM suggested that after injection molding, smaller spherullites, and crystals with higher perfection had formed in the specimens of PP‐A. The resulting of tensile properties of PP‐A were found to be better than that of PP‐B. The results of morphology evolution by SEM observation and 2D‐WAXD showed that PP‐A is more likely to occur interspherulite deformation and can disperse the tensile stress more efficiently, and therefore, its crystal structure can withstand a greater force when tensile stress is applied. On the other hand, PP‐B has larger spherulites and boundaries, and low perfection of lamellaes, and the intraspherulte deformation tend to take place. It is easier for the crystal of PP‐B to be broken up and reoriented along the tensile direction. Copyright © 2014 John Wiley & Sons, Ltd.     

Prometabolites of 5‐Diphospho‐myo‐inositol Pentakisphosphate

Diphospho‐myo‐inositol phosphates (PP‐InsP_y) are an important class of cellular messengers. Thus far, no method for the transport of PP‐InsP_y into living cells is available. Owing to their high negative charge density, PP‐InsP_y will not cross the cell membrane. A strategy to circumvent this issue involves the generation of precursors in which the negative charges are masked with biolabile groups. A PP‐InsP_y prometabolite would require twelve to thirteen biolabile groups, which need to be cleaved by cellular enzymes to release the parent molecules. Such densely modified prometabolites of phosphate esters and anhydrides have never been reported to date. This study discloses the synthesis of such agents and an analysis of their metabolism in tissue homogenates by gel electrophoresis. The acetoxybenzyl‐protected system is capable of releasing 5‐PP‐InsP₅ in mammalian cell/tissue homogenates within a few minutes and can be used to release 5‐PP‐InsP₅ inside cells. These molecules will serve as a platform for the development of fundamental tools required to study PP‐InsP_y physiology.     

Synthesis of 1‐C‐Glycoside‐Linked Lipid II Analogues Toward Bacterial Transglycosylase Inhibition

Preparation of Lipid II analogues containing an enzymatically uncleavable 1‐C‐glycoside linkage between the disaccharide moiety and the pyrophosphate‐ or pyrophosphonate‐lipid moiety is described. The synthesis of a common 1‐C‐vinyl disaccharide intermediate has been developed that allows easy preparation of both an elongated sugar‐phosphate bond and a sugar‐phosphonate moiety, which are coupled with the polyprenyl phosphate to give the desired molecules. Inhibition studies show how a subtle structural modification results in dramatically different potency toward bacterial transglycosylase (TGase), and the results identify Lipid II‐C‐O‐PP (IC₅₀=25 μM ) as a potential TGase inhibitor.     

Polymer ferroelectret based on polypropylene foam: piezoelectric properties prediction using dynamic mechanical analysis

Thin polypropylene (PP) foam films were produced by continuous extrusion using supercritical nitrogen (N₂) and then charged via corona discharge. The samples were characterized by dynamic mechanical analysis as a simple method to predict the piezoelectric properties of the cellular PP obtained. The results were then related to morphological analysis based on scanning electron microscopy and mechanical properties in tension. The results showed that the presence of a nucleating agent (CaCO₃) substantially improved the morphology (in terms of cell size and cell density) of the produced foam. Also, an optimization of the extrusion (screw design, temperature profile, blowing agent, and nucleating agent content) and post‐extrusion (calendering temperature and speed) conditions led to the development of a stretched eye‐like cellular structure with uniform cell size distribution. This morphology produced higher storage and loss moduli in the machine (longitudinal) direction than for the transverse direction, as well as higher piezoelectric properties. The morphological and mechanical results showed that higher cell aspect ratio led to lower Young's modulus, which is suitable to achieve higher piezoelectric properties. Finally, the best quasi‐static piezoelectric d₃₃ coefficient was 550 pC/N for a cellular PP ferroelectret having a uniform eye‐like cellular structure using N₂ as the ionizing gas inside the cells, while the highest value was only 250 pC/N when air was used. Hence, the value of d₃₃ can be improved by more than 100% just by replacing air with N₂ as the ionizing gas. Copyright © 2016 John Wiley & Sons, Ltd.     

Polypropylene/polyperoxide interaction in the melt phase

This paper deals with the effect of polyperoxide on the properties and characteristics of PP. Interaction of PP with polyperoxide was carried out by their mixing in melt phase applying co‐rotating twin screw machine. The analysis of melting/crystallisation processes was performed using DSC. Influence of polyperoxide concentration and mixing condition (temperature, residence time) on PP properties was investigated via determination of PP molecular weight. Mechanism of the PP degradation and decomposition of peroxide groups was judged by thermogravimetric analysis, FT‐IR‐, and NMR‐spectroscopy. It was observed that decomposition of peroxide groups brings about degradation of polypropylene. However, simultaneously some part of short, chains of PP are grafted onto macrochain of the polyperoxide decomposed.     

Oriented (Local) Electric Fields Drive the Millionfold Enhancement of the H‐Abstraction Catalysis Observed for Synthetic Metalloenzyme Analogues

This contribution follows the recent remarkable catalysis observed by Groves et al. in hydrogen‐abstraction reactions by a) an oxoferryl porphyrin radical‐cation complex [Por^?+Fe^IV(O)L_ax] and b) a hydroxoiron porphyrazine ferric complex [PyPzFe^III(OH)L_ax], both of which involve positively charged substituents on the outer circumference of the respective macrocyclic ligands. These charge‐coronated complexes are analogues of the biologically important Compound I (Cpd I) and synthetic hydroxoferric species, respectively. We demonstrate that the observed enhancement of the H‐abstraction catalysis for these systems is a purely electrostatic effect, elicited by the local charges embedded on the peripheries of the respective macrocyclic ligands. Our findings provide new insights into how electrostatics can be employed to tune the catalytic activity of metalloenzymes and can thus contribute to the future design of new and highly efficient hydrogen‐abstraction catalysts.     

Polypropylene nanocomposites with various functionalized‐multiwalled nanotubes: thermomechanical properties,morphology, gas permeation,and optical transparency

Polypropylene (PP) nanocomposites with three different functionalized‐multiwalled nanotubes (F‐MWNTs) are compared in terms of their thermomechanical properties, morphology, oxygen permeability, and optical transparency. The F‐MWNTs dodecanol‐MWNT, dodecylamine‐MWNT, and 1,1,1,3,3,3‐hexafluoro‐2‐phenyl‐2‐propanol‐MWNT were combined with PP to produce hybrid films. The variations of their properties with the matrix polymer F‐MWNT content are discussed. Transmission electron microscopy photographs show that most of the F‐MWNTs are dispersed homogeneously in the matrix polymer on the nanoscale, although some agglomerated F‐MWNT particles are formed. Even composites with low F‐MWNT contents (≤3 wt %) exhibit much better thermomechanical values than pure PP. The gas permeability of the hybrids was found to decrease linearly with increases in the F‐MWNT content of the PP matrix. © 2010 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys, 2010     

Polypropylene/montmorillonite nanocomposites toughened with SEBS‐g‐MA: Structure–property relationship

Polypropylene (PP)/organo‐montmorillonite (Org‐MMT) nanocomposites toughened with maleated styrene‐ethylene‐butylene‐styrene (SEBS‐g‐MA) were prepared via melt compounding. The structure, mechanical properties, and dynamic mechanical properties of PP/SEBS‐g‐MA blends and their nanocomposites were investigated by X‐ray diffraction (XRD), polarizing optical microscopy (POM), tensile, and impact tests. XRD traces showed that Org‐MMT promoted the formation of β‐phase PP. The degree of crystallinity of PP/SEBS‐g‐MA blends and their nanocomposites were determined from the wide angle X‐ray diffraction via profile fitting method. POM experiments revealed that Org‐MMT particles served as nucleating sites, resulting in a decrease of the spherulite size. The essential work of fracture approach was used to evaluate the tensile fracture toughness of the nanocomposites toughened with elastomer. © 2005 Wiley Periodicals, Inc. J Polym Sci Part B: Polym Phys 43: 3112–3126, 2005     

A Tri‐copper(II) Complex Displaying DNA‐Cleaving Properties and Antiproliferative Activity against Cancer Cells

A new disubstituted terpyridine ligand and the corresponding tri‐copper(II) complex have been prepared and characterised. The binding affinity and binding mode of this tri‐copper complex (as well as the previously reported mono‐ and di‐copper analogues) towards duplex DNA were determined by using UV/Vis spectroscopic titrations and fluorescent indicator displacement (FID) assays. These studies showed the three complexes to bind moderately (in the order of 10⁴ M ^?1) to duplex DNA (ct‐DNA and a 26‐mer sequence). Furthermore, the number of copper centres and the nature of the substituents were found to play a significant role in defining the binding mode (intercalative or groove binding). The nuclease potential of the three complexes was investigated by using circular plasmid DNA as a substrate and analysing the products by agarose‐gel electrophoresis. The cleaving activity was found to be dependent on the number of copper centres present (cleaving potency was in the order: tri‐copper>di‐copper>mono‐copper). Interestingly, the tri‐copper complex was able to cleave DNA without the need of external co‐reductants. As this complex displayed the most promising nuclease properties, cell‐based studies were carried out to establish if there was a direct link between DNA cleavage and cellular toxicity. The tri‐copper complex displayed high cytotoxicity against four cancer cell lines. Of particular interest was that it displayed high cytotoxicity against the cisplatin‐resistant MOLT‐4 leukaemia cell line. Cellular uptake studies showed that the tri‐copper complex was able to enter the cell and more importantly localise in the nucleus. Immunoblotting analysis (used to monitor changes in protein levels related to the DNA damage response pathway) and DNA‐flow cytometric studies suggested that this tri‐copper(II) complex is able to induce cellular DNA damage.     

Mechanical properties of polypropylene (PP) + high‐density polyethylene (HDPE) binary blends: Non‐isothermal degradation kinetics of PP + HDPE (80/20) Blends

In this study, the mechanical properties and non‐isothermal degradation kinetics of polypropylene (PP), high‐density polyethylene (HDPE) with dilauroyl peroxide and their blends in different mixture ratios were investigated. The effects of adding dilauroyl peroxide (0–0.20 wt%) on the mechanical and thermal properties of PP + HDPE blends have been studied. On the other hand, the kinetics of the thermal degradation and thermal oxidative degradation of PP + HDPE (80/20 wt%) blends were studied in different atmospheres, to analyze their thermal stability. The kinetic and thermodynamic parameters such as the activation energy, E_a, the pre‐exponential factor, A, the reaction order, n, the entropy change, the enthalpy change, and the free energies of activated complex related to PP, HDPE, and blend systems were calculated by means of the several methods on the basis of the single heating rate. A computer program was developed for automatically processing the data to estimate the reaction parameters by using different models. Most appropriate method was determined for each decomposition step according to the least‐squares linear regression. Copyright © 2013 John Wiley & Sons, Ltd.