(±)-Polysiphenol and Other Analogues via Symmetrical Intermolecular Dimerizations: A Synthetic, Spectroscopic, Structural, and Computational Study.

We report an improved total synthesis of 4,5-dibromo-9,10-dihydrophenanthrene-2,3,6,7-tetraol, (±)-polysiphenol, via intermolecular McMurray dimerization of 5-bromovanillin and subsequent intramolecular oxidative coupling as the key steps. The synthetic route is applicable to 4,5-dichloro- and 4,5-difluoro-halologues (as well as a 4,5-dialkyl-analogue). Distinctive AA'BB' multiplets in their 1H NMR spectra for the dimethylene bridges of the dibromo and dichloro compounds reveal them to be room-temperature stable atropisomers, while for the difluoro compound they present as a singlet. X-ray crystal structure determinations of their tetramethylated synthetic precursors show atropisomeric twist angles of 48°, 46°, and 32°, respectively, with the former representing the largest yet observed in any 4,5-disubstituted-9,10-dihydrophenanthrene. DFT computational studies reveal an unprecedented two-stage atropisomeric interconversion process involving time-independent asynchronous rotations of the dimethylene bridge and the biaryl axis for halologues containing chlorine or bromine, but a more synchronous rotation for the difluoro analogue.

A Novel High-Throughput Assay Reveals That the Temperature Induced Increases in Transphosphatidylation of Phospholipase D Are Dependent on the Alcohol Acceptor Concentration.

Phospholipase D reacts with alcohols or water, transphosphatidylating or hydrolysing lipids such as phosphatidylcholine, generating phosphatidylalcohols or phosphatidic acid, respectively. The enzyme has been employed in many applications making use of the transphosphatidylation reaction and the enzyme's tolerance for organic solvents in order to synthesize natural and artificial phospholipids. Yet, its catalytic properties with respect to the transphosphatidylation reaction are not well understood. Here, we introduce a novel high-throughput assay, making use of 96-well plates, that employs Fluorescamine for the detection of transphosphatidylated amino alcohols. This assay allowed to monitor the KM and VMax at different temperatures, revealing that the former will be elevated by the temperature, while the latter is increased by a combination of both temperature and alcohol acceptor concentration being elevated, suggesting that increase in temperature may open up a new binding site for the alcohol acceptor.

Design, synthesis and evaluation of a tripodal receptor for phosphatidylinositol phosphates.

Phosphatidylinositol phosphates (PIPs) are membrane phospholipids that play crucial roles in a wide range of cellular processes. Their function is dictated by the number and positions of the phosphate groups in the inositol ring (with seven different PIPs being active in the cell). Therefore, there is significant interest in developing small-molecule receptors that can bind selectively to these species and in doing so affect their cellular function or be the basis for molecular probes. However, to date there are very few examples of such molecular receptors. Towards this aim, herein we report a novel tripodal molecule that acts as receptor for mono- and bis-phosphorylated PIPs in a cell free environment. To assess their affinity to PIPs we have developed a new cell free assay based on the ability of the receptor to prevent alkaline phosphatase from hydrolysing these substrates. The new receptor displays selectivity towards two out of the seven PIPs, namely PI(3)P and PI(3,4)P2. To rationalise these results, a DFT computational study was performed which corroborated the experimental results and provided insight into the host-guest binding mode.

Rapid glycosyl-inositol-phospho-ceramide fingerprint from filamentous fungal pathogens using the MALDI Biotyper Sirius system.

RATIONALE: Glycosyl-inositol-phospho-ceramides (GIPCs) or glycosylphosphatidylinositol-anchored fungal polysaccharides are known to be major lipids in plant and fungal plasma membranes and to play an important role in stress adaption. However, their analysis remains challenging due to the several steps involved for their extractions and purifications prior to mass spectrometric analysis. To address this challenge, we developed a rapid and sensitive method to identify GIPCs from the four common fungal plant pathogens Botrytis cinerea, Fusarium graminearium, Neurospora crassa and Ustilago maydis. METHODS: Fungal plant pathogens were cultured, harvested, heat-inactivated and washed three times with double-distilled water. Intact fungi were deposited on a matrix-assisted laser desorption ionization (MALDI) target plate, mixed with the matrix consisting of a 9:1 mixture of 2,5-dihydroxybenzoic acid and 2-hydroxy-5-methoxybenzoic acid solubilized at 10 mg/mL in chloroform-methanol (9:1 v/v) and analyzed using a Bruker MALDI Biotyper Sirius system in the linear negative ion mode. Mass spectra were acquired from m/z 700 to 2000. RESULTS: MALDI time-of-flight (TOF) mass spectrometric analysis of cultured fungi showed clear signature of GIPCs in B. cinerea, F. graminearium, N. crassa and U. maydis. CONCLUSIONS: We have demonstrated that routine MALDI-TOF in the linear negative ion mode combined with an apolar solvent system to solubilize the matrix is applicable to the detection of filamentous fungal GIPCs.

A tri-functional vanadium(iv) complex to detect cysteine oxidation.

The development of effective molecular probes to detect and image the levels of oxidative stress in cells remains a challenge. Herein we report the design, synthesis and preliminary biological evaluation of a novel optical probe to monitor oxidation of thiol groups in cysteine-based phosphatases (CBPs). Following orthogonal protecting approaches we synthesised a new vanadyl complex designed to bind to CBPs. This complex is functionalised with a well-known dimedone derivative (to covalently trap sulfenic acids, SOHs) and a coumarin-based fluorophore for optical visualization. We show that this new probe efficiently binds to a range of phosphatases in vitro with nanomolar affinity. Moreover, preliminary flow cytometry and microscopy studies in live HCT116 cells show that this probe can successfully image cellular levels of sulfenic acids - one of the species resulting from protein oxidative damage.

A lipophilic copper(ii) complex as an optical probe for intracellular detection of NO.

A new chemical sensor for cellular imaging of NO is presented. This cell-permeable probe is based on a complex where copper(ii) is coordinated to a tridentate ligand substituted with a fluorophore (NBD) and an octyl group. The fluorescence response of this complex towards a range of reactive species (namely NO, NO2-, NO3-, H2O2, ClO-, O2- and ONOO-) has been studied in vitro showing that the probe is highly selective for NO. The probe is readily taken up by cells and is able to image the cellular concentrations of NO.

Vanadyl complexes with dansyl-labelled di-picolinic acid ligands: synthesis, phosphatase inhibition activity and cellular uptake studies.

Vanadium complexes have been previously utilised as potent inhibitors of cysteine based phosphatases (CBPs). Herein, we present the synthesis and characterisation of two new fluorescently labelled vanadyl complexes (14 and 15) with bridged di-picolinic acid ligands. These compounds differ significantly from previous vanadyl complexes with phosphatase inhibition properties in that the metal-chelating part is a single tetradentate unit, which should afford greater stability and scope for synthetic elaboration than the earlier complexes. These new complexes inhibit a selection of cysteine based phosphatases (CBPs) in the nM range with some selectivity. Fluorescence spectroscopic studies (including fluorescence anisotropy) were carried out to demonstrate that the complexes are not simply acting as vanadyl delivery vehicles but they interact with the proteins. Finally, we present preliminary fluorescence microscopy studies to demonstrate that the complexes are cell permeable and localise throughout the cytoplasm of NIH3T3 cells.

Reversible oxidation of phosphatase and tensin homolog (PTEN) alters its interactions with signaling and regulatory proteins.

Phosphatase and tensin homolog (PTEN) is involved in a number of different cellular processes including metabolism, apoptosis, cell proliferation and survival. It is a redox-sensitive dual-specificity protein phosphatase that acts as a tumor suppressor by negatively regulating the PI3K/Akt pathway. While direct evidence of redox regulation of PTEN downstream signaling has been reported, the effect of PTEN redox status on its protein-protein interactions is poorly understood. PTEN-GST in its reduced and a DTT-reversible H2O2-oxidized form was immobilized on a glutathione-sepharose support and incubated with cell lysate to capture interacting proteins. Captured proteins were analyzed by LC-MSMS and comparatively quantified using label-free methods. 97 Potential protein interactors were identified, including a significant number that are novel. The abundance of fourteen interactors was found to vary significantly with the redox status of PTEN. Altered binding to PTEN was confirmed by affinity pull-down and Western blotting for Prdx1, Trx, and Anxa2, while DDB1 was validated as a novel interactor with unaltered binding. These results suggest that the redox status of PTEN causes a functional variation in the PTEN interactome. The resin capture method developed had distinct advantages in that the redox status of PTEN could be directly controlled and measured.

OCRL1 engages with the F-BAR protein pacsin 2 to promote biogenesis of membrane-trafficking intermediates.

Mutation of the inositol 5-phosphatase OCRL1 causes Lowe syndrome and Dent-2 disease. Loss of OCRL1 function perturbs several cellular processes, including membrane traffic, but the underlying mechanisms remain poorly defined. Here we show that OCRL1 is part of the membrane-trafficking machinery operating at the trans-Golgi network (TGN)/endosome interface. OCRL1 interacts via IPIP27A with the F-BAR protein pacsin 2. OCRL1 and IPIP27A localize to mannose 6-phosphate receptor (MPR)-containing trafficking intermediates, and loss of either protein leads to defective MPR carrier biogenesis at the TGN and endosomes. OCRL1 5-phosphatase activity, which is membrane curvature sensitive, is stimulated by IPIP27A-mediated engagement of OCRL1 with pacsin 2 and promotes scission of MPR-containing carriers. Our data indicate a role for OCRL1, via IPIP27A, in regulating the formation of pacsin 2-dependent trafficking intermediates and reveal a mechanism for coupling PtdIns(4,5)P2 hydrolysis with carrier biogenesis on endomembranes.

Targeting PTEN using small molecule inhibitors.

PTEN (phosphatase and tensin homologue deleted on chromosome 10) is well known as a tumour suppressor. It's PI(3,4,5)P3 lipid phosphatase activity is an important counteracting mechanism in PI 3-kinase (phosphoinositide 3-kinase) signalling. Furthermore, PTEN lies upstream of Akt kinase, a key enzyme in insulin signalling regulating glucose uptake and cell growth. Therefore, PTEN has recently gained attention as a valuable drug target for the treatment of diabetes, stroke, cardiac infarct and fertility. This review summarizes the use of small molecules as PTEN inhibitors. Currently available methodologies and techniques for accessing PTEN inhibition in vitro and in cellulo will be discussed.

Synthesis of unsaturated phosphatidylinositol 4-phosphates and the effects of substrate unsaturation on SopB phosphatase activity.

In this paper evidence is presented that the fatty acid component of an inositide substrate affects the kinetic parameters of the lipid phosphatase Salmonella Outer Protein B (SopB). A succinct route was used to prepare the naturally occurring enantiomer of phosphatidylinositol 4-phosphate (PI-4-P) with saturated, as well as singly, triply and quadruply unsaturated, fatty acid esters, in four stages: (1) The enantiomers of 2,3:5,6-O-dicyclohexylidene-myo-inositol were resolved by crystallisation of their di(acetylmandelate) diastereoisomers. (2) The resulting diol was phosphorylated regio-selectively exclusively on the 1-O using the new reagent tri(2-cyanoethyl)phosphite. (3) With the 4-OH still unprotected, the glyceride was coupled using phosphate tri-ester methodology. (4) A final phosphorylation of the 4-O, followed by global deprotection under basic then acidic conditions, provided PI-4-P bearing a range of sn-1-stearoyl, sn-2-stearoyl, -oleoyl, -γ-linolenoyl and arachidonoyl, glycerides. Enzymological studies showed that the introduction of cis-unsaturated bonds has a measurable influence on the activity (relative Vmax) of SopB. Mono-unsaturated PI-4-P exhibited a five-fold higher activity, with a two-fold higher KM, over the saturated substrate, when presented in DOPC vesicles. Poly-unsaturated PI-4-P showed little further change with respect to the singly unsaturated species. This result, coupled with our previous report that saturated PI-4-P has much higher stored curvature elastic stress than PI, supports the hypothesis that the activity of inositide phosphatase SopB has a physical role in vivo.

Highly efficient and selective phosphorylation of amino acid derivatives and polyols catalysed by 2-aryl-4-(dimethylamino)pyridine-N-oxides--towards kinase-like reactivity.

The chemoselective phosphorylation of hydroxyl containing amino acid derivatives and polyols by phosphoryl chlorides catalyzed by 2-aryl-4-(dimethylamino)pyridine-N-oxides is described.

Chemical intervention tools to probe phosphoinositide-dependent signalling.

Chemical intervention tools have been beneficial to many investigations elucidating signalling networks and interactions. The present review summarizes the current status of chemical tools to probe phosphoinositide metabolism and signalling. In particular, phosphoinositide-targeting tools are compared with protein-targeting tools with respect to their unique advantages and possible applications.

Isolation and kinetic characterisation of hydrophobically distinct populations of form I Rubisco.

BACKGROUND: Rubisco (Ribulose-1,5-bisphosphate carboxylase/oxygenase) is a Calvin Cycle enzyme involved in CO2 assimilation. It is thought to be a major cause of photosynthetic inefficiency, suffering from both a slow catalytic rate and lack of specificity due to a competing reaction with oxygen. Revealing and understanding the engineering rules that dictate Rubisco's activity could have a significant impact on photosynthetic efficiency and crop yield. RESULTS: This paper describes the purification and characterisation of a number of hydrophobically distinct populations of Rubisco from both Spinacia oleracea and Brassica oleracea extracts. The populations were obtained using a novel and rapid purification protocol that employs hydrophobic interaction chromatography (HIC) as a form I Rubisco enrichment procedure, resulting in distinct Rubisco populations of expected enzymatic activities, high purities and integrity. CONCLUSIONS: We demonstrate here that HIC can be employed to isolate form I Rubisco with purities and activities comparable to those obtained via ion exchange chromatography (IEC). Interestingly, and in contrast to other published purification methods, HIC resulted in the isolation of a number of hydrophobically distinct Rubisco populations. Our findings reveal a so far unaccounted diversity in the hydrophobic properties within form 1 Rubisco. By employing HIC to isolate and characterise Spinacia oleracea and Brassica oleracea, we show that the presence of these distinct Rubisco populations is not species specific, and we report for the first time the kinetic properties of Rubisco from Brassica oleracea extracts. These observations may aid future studies concerning Rubisco's structural and functional properties.

A unified nomenclature and amino acid numbering for human PTEN.

The tumor suppressor PTEN is a major brake for cell transformation, mainly due to its phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] phosphatase activity that directly counteracts the oncogenicity of phosphoinositide 3-kinase (PI3K). PTEN mutations are frequent in tumors and in the germ line of patients with tumor predisposition or with neurological or cognitive disorders, which makes the PTEN gene and protein a major focus of interest in current biomedical research. After almost two decades of intense investigation on the 403-residue-long PTEN protein, a previously uncharacterized form of PTEN has been discovered that contains 173 amino-terminal extra amino acids, as a result of an alternate translation initiation site. To facilitate research in the field and to avoid ambiguities in the naming and identification of PTEN amino acids from publications and databases, we propose here a unifying nomenclature and amino acid numbering for this longer form of PTEN.

Protocell design through modular compartmentalization.

De novo synthetic biological design has the potential to significantly impact upon applications such as energy generation and nanofabrication. Current designs for constructing organisms from component parts are typically limited in scope, as they utilize a cut-and-paste ideology to create simple stepwise engineered protein-signalling pathways. We propose the addition of a new design element that segregates components into lipid-bound 'proto-organelles', which are interfaced with response elements and housed within a synthetic protocell. This design is inspired by living cells, which utilize multiple types of signalling molecules to facilitate communication between isolated compartments. This paper presents our design and validation of the components required for a simple multi-compartment protocell machine, for coupling a light transducer to a gene expression system. This represents a general design concept for the compartmentalization of different types of artificial cellular machinery and the utilization of non-protein signal molecules for signal transduction.

Molecular recognition with boronic acids-applications in chemical biology.

Small molecules have long been used for the selective recognition of a wide range of analytes. The ability of these chemical receptors to recognise and bind to specific targets mimics certain biological processes (such as protein-substrate interactions) and has therefore attracted recent interest. Due to the abundance of biological molecules possessing polyhydroxy motifs, boronic acids-which form five-membered boronate esters with diols-have become increasingly popular in the synthesis of small chemical receptors. Their targets include biological materials and natural products including phosphatidylinositol bisphosphate, saccharides and polysaccharides, nucleic acids, metal ions and the neurotransmitter dopamine. This review will focus on the many ways in which small chemical receptors based on boronic acids have been used as biochemical tools for various purposes, including sensing and detection of analytes, interference in signalling pathways, enzyme inhibition and cell delivery systems. The most recent developments in each area will be highlighted.

Alternative synthetic tools to phospho-specific antibodies for phosphoproteome analysis: progress and prospects.

Signal transduction cascades in living systems are often controlled via post-translational phosphorylation and dephosphorylation of proteins. These processes are catalyzed in vivo by kinase and phosphatase enzymes, which consequently play an important role in many disease states, including cancer and immune system disorders. Current techniques for studying the phosphoproteome (isotopic labeling, chromatographic techniques, and phosphospecific antibodies), although undoubtedly very powerful, have yet to provide a generic tool for phosphoproteomic analysis despite the widespread utility such a technique would have. The use of small molecule organic catalysts that can promote selective phosphate esterification could provide a useful alternative to current state-of-the-art techniques for use in, e.g., the labeling and pull-down of phosphorylated proteins. This report reviews current techniques used for phosphoproteomic analysis and the recent use of small molecule peptide-based catalysts in phosphorylation reactions, indicating possible future applications for this type of catalyst as synthetic alternatives to phosphospecific antibodies for phosphoproteome analysis.

Acute manipulation of diacylglycerol reveals roles in nuclear envelope assembly & endoplasmic reticulum morphology.

The functions and morphology of cellular membranes are intimately related and depend not only on their protein content but also on the repertoire of lipids that comprise them. In the absence of in vivo data on lipid asymmetry in endomembranes, it has been argued that motors, scaffolding proteins or integral membrane proteins rather than non-lamellar bilayer lipids such as diacylglycerol (DAG), are responsible for shaping of organelles, local membrane curvature and fusion. The effects of direct alteration of levels of such lipids remain predominantly uninvestigated. Diacylglycerol (DAG) is a well documented second messenger. Here we demonstrate two additional conserved functions of DAG: a structural role in organelle morphology, and a role in localised extreme membrane curvature required for fusion for which proteins alone are insufficient. Acute and inducible DAG depletion results in failure of the nuclear envelope (NE) to reform at mitosis and reorganisation of the ER into multi-lamellar sheets as revealed by correlative light and electron microscopy and 3D reconstructions. Remarkably, depleted cells divide without a complete NE, and unless rescued by 1,2 or 1,3 DAG soon die. Attenuation of DAG levels by enzyme microinjection into echinoderm eggs and embryos also results in alterations of ER morphology and nuclear membrane fusion. Our findings demonstrate that DAG is an in vivo modulator of organelle morphology in mammalian and echinoderm cells, indicating a fundamental role conserved across the deuterostome superphylum.