Stabilized phosphatidylinositol-5-phosphate analogues as ligands for the nuclear protein ING2: chemistry, biology, and molecular modeling

The interaction of PtdIns(5)P with the tumor suppressor protein ING2 has been implicated in the regulation of chromatin modification. To enhance the stability of PtdIns(5)P for studies of the biological role in vivo, two phosphatase-resistant moieties were used to replace the labile 5-phosphate. The total asymmetric synthesis of the 5-methylenephosphonate (MP) and 5-phosphothionate (PT) analogues of PtdIns(5)P is described herein, and the resulting metabolically stabilized lipid analogues were evaluated in three ways. First, liposomes containing either the dioleoyl MP or PT analogues bound to recombinant ING2 similar to liposomes containing dipalmitoyl PtdIns(5)P, indicating that the replacement of the hydrolyzable 5-phosphate group does not compromise the binding. Second, the dioleoyl MP and PT PtdIns(5)P analogues were equivalent to dipalmitoyl PtdIns(5)P in augmenting cell death induced by a DNA double-strand break in HT1080 cells. Finally, molecular modeling and docking of the MP or PT analogues to the C-terminus PtdInsP-binding region of ING2 (consisting of a PHD finger and a polybasic region) revealed a number of complementary surface and electrostatic contacts between the lipids and ING2.     

Metabolically stabilized derivatives of phosphatidylinositol 4-phosphate: synthesis and applications

Phosphatidylinositol 4-phosphate (PtdIns(4)P) lipid is an essential component of eukaryotic membranes and a marker of the Golgi complex. Here, we developed metabolically stabilized (ms) analogs of PtdIns(4)P and the inositol 1,4-bisphosphate (IP(2)) head group derivative and demonstrated that these compounds can substitute the natural lipid fully retaining its physiological activities. The methylenephosphonate (MP) and phosphorothioate (PT) analogs of PtdIns(4)P and the aminohexyl (AH)-IP(2) probe are recognized by the PtdIns(4)P-specific PH domain of four phosphate adaptor protein 1 (FAPP1). Binding of FAPP1 to the PtdIns(4)P derivatives stimulates insertion of the PH domain into the lipid layers and induces tubulation of membranes. Both ms analogs and IP(2) probes could be invaluable for identifying protein effectors and characterizing PtdIns(4)P-dependent signaling cascades within the trans-Golgi network (TGN).     

Chemical synthesis and molecular recognition of phosphatase-resistant analogues of phosphatidylinositol-3-phosphate

The remodeling of phosphatidylinositol polyphosphates in cellular membranes by phosphatases and kinases orchestrates the signaling by these lipids in space and time. To provide chemical tools to study the changes in cell physiology mediated by these lipids, three new metabolically stabilized (ms) analogues of phosphatidylinositol-3-phosphate (PtdIns(3)P) were synthesized. We describe herein the total asymmetric synthesis of 3-methylphosphonate, 3-(monofluoromethyl)phosphonate and 3-phosphorothioate analogues of PtdIns(3)P. From differentially protected D-myo-inositol key intermediates, a versatile phosphoramidite reagent was employed in the synthesis of PtdIns(3)P analogues with diacylglyceryl moieties containing dioleoyl, dipalmitoyl, and dibutyryl chains. In addition, we introduce a new phosphorylation reagent, (monofluoromethyl)phosphonyl chloride, which has general applications for the preparation of "pKa-matched" monofluorophosphonates. These ms-PtdIns(3)P analogues exhibited reduced binding activities with 15N-labeled FYVE and PX domains, as significant 1H and 15N chemical shift changes in the FYVE domain were induced by titrating ms-PtdIns(3)P analogues into membrane-mimetic dodecylphosphocholine micelles. In addition, the PtdIns(3)P analogues with dioleoyl and dipalmitoyl chains were substrates for the 5-kinase enzyme PIKfyve; the corresponding phosphorylated ms-PI(3,5)P2 products were detected by radio-TLC analysis.     

Synthesis and biological activity of PTEN-resistant analogues of phosphatidylinositol 3,4,5-trisphosphate

The activation of phosphatidylinositol 3-kinase (PI 3-K) and subsequent production of PtdIns(3,4,5)P3 launches a signal transduction cascade that impinges on a plethora of downstream effects on cell physiology. Control of PI 3-K and PtdIns(3,4,5)P3 levels is an important therapeutic target in treatments for allergy, inflammation, cardiovascular, and malignant human diseases. We designed metabolically stabilized, that is, phosphatase resistant, analogues of PtdIns(3,4,5)P3 as probes for long-lived potential agonists or potential antagonists for cellular events mediated by PtdIns(3,4,5)P3. In particular, two types of analogues were prepared containing phosphomimetics that would be selectively resistant to the lipid 3-phosphatase PTEN. The total asymmetric synthesis of the 3-phosphorothioate-PtdIns(3,4,5)P3 and 3-methylenephosphonate-PtdIns(3,4,5)P3 analogues is described. These two analogues showed differential binding to PtdIns(3,4,5)P3 binding modules, and both were potential long-lived activators that mimicked insulin action in sodium transport in A6 cells.     

Synthesis and biological activity of phospholipase C-resistant analogues of phosphatidylinositol 4,5-bisphosphate

The membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) is an important regulator in cell physiology. Hydrolysis of PtdIns(4,5)P2 by phospholipase C (PLC) releases two second messengers, Ins(1,4,5)P3 and diacylglycerol. To dissect the effects of PtdIns(4,5)P2 from those resulting from PLC-generated signals, a metabolically stabilized analogue of PtdIns(4,5)P2 was required. Two analogues were designed in which the scissile O-P bond was replaced with a C-P bond that could not be hydrolyzed by PLC activity. Herein we describe the asymmetric total synthesis of the first metabolically stabilized phospholipase C-resistant analogues of PtdIns(4,5)P2. The key transformation was a Pd(0)-catalyzed coupling of a H-phosphite with a vinyl bromide to form the desired C-P linkage. The phosphonate analogues of PtdIns(4,5)P2 were found to be effective in restoring the sensitivity of the TRPM4 channel to Ca2+ activation.     

Quantitative analysis of inositol lipids and inositol phosphates in synaptosomes and microvessels by column chromatography: comparison of the mass analysis and the radiolabelling methods.

Chromatographic methods that measure both the mass and the radiolabelling of various inositol lipids and inositol phosphates in tissues have been developed. The mass of phosphatidylinositol (PtdIns), phosphatidylinositol-4-monophosphate [PtdIns(4)P] and phosphatidylinositol-4,5-bisphosphate [PtdIns(4,5)P2] was quantitated by measuring the inorganic phosphate, whereas inositol monophosphate (IP), inositol bisphosphate (IP2), inositol trisphosphate (IP3) and inositol tetrakisphosphate (IP4) were quantitated by using an enzymic method. The radiolabelling of various inositol lipids and inositol phosphates was determined by incubating the tissue samples with [3H]myo-inositol, separating individual inositol lipids and inositol phosphates, and measuring the radioactivity in each compound. Although the mass analysis method was sensitive enough to measure low levels of inositol lipids or inositol phosphates, the method was laborious and time-consuming. Compared with the enzymic method, the radiolabelling method was simple and fast, but it gave variable results. This study demonstrated differences in inositol lipid and inositol phosphate levels by radiolabelling and mass measurements, and agonist-stimulated phosphatidylinositol turnover of synaptosomes versus the blood-brain barrier as represented by microvessels. Although the mass of PtdIns, PtdIns(4)P and PtdIns(4,5)P2 was comparable in synaptosomes and microvessels, the incorporation of [3H]myo-inositol into phosphorylated PtdIns in microvessels was less than that in synaptosomes.     

Rapid and efficient routes to phosphatidylinositol 3,4,5-trisphosphates via myo-inositol orthobenzoate

Efficient routes to two phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P₃] analogues with different acyl chains have been developed by using cheaply available myo-inositol as the starting material. The high yield of the orthobenzoate derivative, preferential formation of the required protected inositol diastereomer in its desymmetrization and ease of separation make the synthesis expedient, economical and high yielding. Due to the inherent problem of racemization of diacylglycerol (DAG), the synthesis of phosphatidylinositol phosphates [PIPns] with unambiguous stereochemical purity has always been difficult. Our methodology excludes the possibility of racemization in the DAG unit and thus provides access to PtdIns(3,4,5)P₃ of high optical purity. Since the acyl functionalities are introduced last, the methodology reported is amenable to the synthesis of PtdIns(3,4,5)P₃ with any acyl chain (or even a library of analogues).     

Understanding the relative affinity and specificity of the pleckstrin homology domain of protein kinase B for inositol phosphates

Protein kinase B (PKB) is a serine/threonine kinase that plays a key role in the phosphoinositide 3-kinase (PI3K) pathway-one of the most frequently activated proliferation pathways in cancer. In this pathway, PKB is recruited to the plasma membrane by direct interaction of its pleckstrin homology (PH) domain with the inositol phosphate head-group of phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] or phosphatidylinositol 3,4-bisphosphate [PtdIns(3,4)P(2)]. This recruitment is a critical stage in the activation of PKB, whose downstream effectors play important roles in cell survival, proliferation and growth. It is therefore of great interest to understand PKB's mode of binding, as well as its specificity and affinity for different phosphoinositides. We have used a total of 3 μs of molecular dynamics (MD) simulations to better understand the interactions of the PKB PH domain with the inositol phosphate head-groups of phosphoinositides involved in the PI3K pathway. Our computational models successfully mirror PKB's in vivo selectivity for 3-phosphorylated phosphoinositides. Furthermore, the models also help to rationalize unexpected in vitro data in which inositol 1,4,5-trisphosphate [Ins(1,4,5)P(3)] binds with a relatively high affinity to the PKB PH domain, despite its parent lipid phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P(2)] being known not to bind in vivo. With the support of computational simulations, we propose that when not bonded to a phosphatidate tail Ins(1,4,5)P(3) binds in an orientation in which its inositol ring is flipped with respect to the 3-phosphorylated inositol phosphate ligands and its parent lipid.     

Synthesis of Photoactivatable 1,2-O-Diacyl-sn-glycerol Derivatives of 1-L-Phosphatidyl-D-myo-inositol 4,5-Bisphosphate (PtdInsP(2)) and 3,4,5-Trisphosphate (PtdInsP(3))

Photoactivatable analogues of 1-L-phosphatidyl-D-myo-inositol 4,5-bisphosphate (PtdIns(4,5)P(2) or PtdInsP(2)) and the corresponding 3,4,5-trisphosphate (PtdIns(3,4,5)P(3) or PtdInsP(3)) were prepared from the two chiral precursors, methyl alpha-D-glucopyranoside and 1,2-isopropylidene-sn-glycerol. Two key synthetic transformations included the Ferrier rearrangement reaction to construct the optically-pure inositol skeleton and the sequential acylation of the primary and secondary hydroxyl groups on the glycerol derivatives. The sn-1-O-(6-aminohexanoyl) PtdInsP(2) and PtdInsP(3) derivatives were further modified to contain benzophenone photophores in unlabeled and high specific activity tritium-labeled forms.     

Determination of airborne methyl isocyanate as dibutylamine or 1-(2-methoxyphenyl)piperazine derivatives by liquid and gas chromatography

The usefulness of a glass fibre filter method to collect airborne methyl isocyanate (MIC) was studied in laboratory experiments and in a workplace during manufacture of mineral wool insulation material. Filter collection was based on derivatisation in situ with 1-(2-methoxyphenyl)piperazine (2MP). 2MP impinger sampling was also evaluated in the workplace. Impinger sampling with dibutylamine (DBA) was used as an independent method. The samples were analysed by liquid and gas chromatography using various detection techniques: mass spectrometry, ultraviolet and electrochemical detection (LC-MS, LC-UV, LC-EchD and GC-MS). The sampling efficiency of 2MP filters for MIC varied with the origin of the glass fibre filter. Two Whatman filters (diameter 25 mm) with altogether 21 mumol of 2MP collected 100% of 9.8 micrograms of MIC during 30 min at an airflow rate of 1 l min-1. The workplace measurements were performed at two concentration levels, 0.003 and 0.09 mg m-3. The theoretical amounts of derivatisation reagent were 42 mumol (2MP filter), 52 mumol (2MP impinger) and 100 mumol (DBA). MIC concentrations were 20% lower by the 2MP methods compared with the DBA method (statistically significant difference). Breakthrough was 6% for the DBA method and 9% for the 2MP impinger method. To trap both MIC and isocyanic acid, which was also present in the workplace samples, a tenfold molar amount of 2MP reagent was used. The precision of sample preparation, expressed as relative standard deviation, was 3.5% (0.17 microgram ml-1, n = 6). The precision of sampling in the workplace was 15% (0.002 mg m-3, n = 6). The limit of quantification was 0.0006 mg m-3 for 30 l of air by the 2MP impinger method and 0.03-0.05 mg m-3 by the 2MP filter method. Hence, airborne MIC can be determined using 2MP as derivatisation reagent. Impinger sampling is preferable when low concentration levels are expected.     

Analysis of vibrational spectra of 3-halo-1-propanols CH(2)XCH(2)CH(2)OH (X is Cl and Br)

The conformational stability and the three rotor internal rotations in 3-chloro- and 3-bromo-1-propanols were investigated by DFT-B3LYP/6-311+G and ab initio MP2/6-311+G, MP3/6-311+G and MP4(SDTQ)//MP3/6-311+G levels of theory. On the calculated potential energy surface twelve distinct minima were located all of which were not predicted to have imaginary frequencies at the B3LYP level of theory. The calculated lowest energy minimum in the potential curves of both molecules was predicted to correspond to the Gauche-gauche-trans (Ggt) conformer in excellent agreement with earlier microwave and electron diffraction results. The equilibrium constants for the conformational interconversion of the two 3-halo-1-propanols were calculated at the B3LYP/6-311+G level of calculation and found to correspond to an equilibrium mixture of about 32% Ggt, 18% Ggg1, 13% Tgt, 8% Tgg and 8% Gtt conformations for 3-chloro-1-propanol and 34% Ggt, 15% Tgt, 13% Ggg1, 9% Tgg and 7% Gtt conformations for 3-bromo-1-propanol at 298.15K. The nature of the high energy conformations was verified by carrying out solvent experiments using formamide ( epsilon=109.5) and MP3 and MP4//MP3 calculations. The vibrational frequencies of each molecule in its three most stable forms were computed at the B3LYP level and complete vibrational assignments were made based on normal coordinate calculations and comparison with experimental data of the molecules.     

Three-phase solvent systems for comprehensive separation of a wide variety of compounds by high-speed counter-current chromatography

Three-phase solvent systems were efficiently utilized for high-speed counter-current chromatography (HSCCC) to separate multiple components with a wide range of hydrophobicity. The compositions of three-phase systems were optimized according to their physical parameters such as volume ratio, viscosity and specific gravity of upper (UP), middle (MP) and lower (LP) phases. The three-phase systems composed of n-hexane-methyl acetate-acetonitrile-water (4:4:3:4, v/v/v/v) was selected for HSCCC separation of a mixture of 15 standard compounds with a wide range in hydrophobicity from beta-carotene to tryptophan. The separation was initiated by filling the column with a mixture of MP and LP both as a stationary phase followed by elution with UP to separate the hydrophobic compounds. Then the mobile phase was switched to MP to elute the moderately hydrophobic compounds, and finally the polar compounds still retained in the column were fractionated by eluting the column with LP. The system successfully resolved all 15 compounds in one-step operation in 70 min.     

A family of ordered mesoporous carbons derived from mesophase pitch using ordered mesoporous silicas as templates

A variety of ordered mesoporous carbons (OMCs) were synthesized using ordered mesoporous silicas (OMSs) as hard templates and the mesophase pitch (MP) as a carbon precursor. The synthesis included the mixing of OMS with MP, the infiltration of OMS with MP at 450–550?°C and the carbonization of MP in OMS/MP composite followed by the dissolution of the OMS template. OMCs with structures of two-dimensional hexagonal arrays of nanorods and three-dimensional arrays of nanospheres were obtained through the replication of silica templates, including large-pore SBA-15, KIT-6, large-pore FDU-12 and SBA-16. In particular, 2-D hexagonal array of carbon nanorods (CMK-3 carbon) with (100) interplanar spacing of ～13 nm as well as an array of carbon nanospheres arranged in the face-centered cubic structure with the unit-cell parameter of 33 nm were successfully prepared. The specific surface areas of the resulting carbons were up to 400 m²/g, and the total pore volumes were up to 0.43 cm³/g, with the highest values achieved when the MP infiltration temperature was 500?°C. The OMCs exhibited narrow mesopore size distributions. As inferred from XRD, the frameworks of OMCs featured semi-graphitic structures even though moderate carbonization temperature (850?°C) was employed.     

Synthesis of hybrid lipid probes: derivatives of phosphatidylethanolamine-extended phosphatidylinositol 4,5-bisphosphate (Pea-PIP(2))

The total asymmetric synthesis of a novel hybrid lipid possessing a 2,3-diacylthreitol backbone, rather than a 1,2-diacylglycerol backbone, is described. The title compound, Pea-PIP(2), possesses a phosphatidylethanolamine (PE) headgroup at the 1-position and a phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) headgroup at the 4-position. Reporters (biotin, fluorophores, spin label) were covalently attached to the free amino group of the PE, such that these reporters were targeted to the lipid-water interface. The diacyl moieties allow incorporation of Pea-PIP(2) into a lipid bilayer, while the PtdIns(4,5)P(2) moiety in the aqueous layer was specifically recognized by PtdIns(4,5)P(2)-specific binding proteins.     

Microwave-assisted extraction of phenolic antioxidants from potato peels

A response surface method was used to optimize the microwave-assisted extraction parameters such as extraction time (t) (min), solvent (methanol) concentration (S) (v/v) and microwave power level (MP) for extraction of antioxidants from potato peels. Max. total phenolics content of 3.94 mg g?1 dry weight (dw) was obtained at S of 67.33%, t of 15 min and a MP of 14.67%. For ascorbic acid (1.44 mg g?1 dw), caffeic acid (1.33 mg g?1 dw), ferulic acid (0.50 mg g?1 dw) max contents were obtained at S of 100%, t of 15 min, and MP of 10%, while the max chlorogenic acid content (1.35 mg g?1 dw) was obtained at S of 100%, t of 5 min, and MP of 10%. The radical scavenging activity of the extract was evaluated by using the DPPH assay and optimum antioxidant activity was obtained at S of 100%, t of 5 min, and MP of 10%.     

Systematically convergent basis sets for explicitly correlated wavefunctions: the atoms H, He, B-Ne, and Al-Ar

Correlation consistent basis sets have been optimized for use with explicitly correlated F12 methods. The new sets, denoted cc-pVnZ-F12 (n=D,T,Q), are similar in size and construction to the standard aug-cc-pVnZ and aug-cc-pV(n+d)Z basis sets, but the new sets are shown in the present work to yield much improved convergence toward the complete basis set limit in MP2-F12/3C calculations on several small molecules involving elements of both the first and second row. For molecules containing only first row atoms, the smallest cc-pVDZ-F12 basis set consistently recovers nearly 99% of the MP2 valence correlation energy when combined with the MP2-F12/3C method. The convergence with basis set for molecules containing second row atoms is slower, but the new DZ basis set still recovers 97%-99% of the frozen core MP2 correlation energy. The accuracy of the new basis sets for relative energetics is demonstrated in benchmark calculations on a set of 15 chemical reactions.     

Effects of pH of fermentation medium on biosynthesis of poly[(3-hydroxybutyrate)-co-(3-mercaptopropionate)] by Wautersia eutropha

A series of P(3HB-co-3MP)s with different 3MP unit content was biosynthesized by the fermentation of W. eutropha in a medium containing sodium gluconate and DTDP as carbon sources at different pH conditions ranging from pH 6.0 to 8.0. The P(3HB-co-3MP) samples were fractioned using the solvent/nonsolvent mixed solvent chloroform/heptane and the comonomer unit composition was investigated. It was found that W. eutropha produces P(3HB-co-3MP)s with extremely different 3MP unit content ranging from 3.6 to 70.0 mol-%, depending on the pH value of the fermentation medium. The copolyester samples produced in mild basic medium have a considerably narrower compositional distribution than the samples from acidic medium. The highest polymer yield was obtained at pH 8.0.DSC diagram for P(3HB-co-3MP)s biosynthesized in different pH medium. [graph: see text] DSC diagram for P(3HB-co-3MP)s biosynthesized in different pH medium.     

Experimental and theoretical studies on the thermal decomposition of heterocyclic nitrosimines

A series of substituted 2-nitrosiminobenzothiazolines (2) were synthesized by the nitrosation of the corresponding 2-iminobenzothiazolines (6). Thermal decomposition of 2a--f and of the seleno analogue 7 in methanol and of 3-methyl-2-nitrosobenzothiazoline (2a) in acetonitrile, 1,4-dioxane, and cyclohexane followed first-order kinetics. The activation parameters for thermal deazetization of 2a were measured in cyclohexane (Delta H(++) = 25.3 +/- 0.5 kcal/mol, Delta S(++) = 1.3 +/- 1.5 eu) and in methanol (Delta H(++) = 22.5 +/- 0.7 kcal/mol, Delta S(++) = -12.9 +/- 2.1 eu). These results indicate a unimolecular decomposition and are consistent with a proposed stepwise mechanism involving cyclization of the nitrosimine followed by loss of N(2). The ground-state conformations of the parent nitrosiminothiazoline (9a) and transition states for rotation around the exocyclic C==N bond, electrocyclic ring closure, and loss of N(2) were calculated using ab initio molecular orbital theory at the MP2/6-31G* level. The calculated gas-phase barrier height for the loss of N(2) from 9a (25.2 kcal/mol, MP4(SDQ, FC)/6-31G*//MP2/6-31G* + ZPE) compares favorably with the experimental barrier for 2a of 25.3 kcal/mol in cyclohexane. The potential energy surface is unusual; the rotational transition state 9a-rot-ts connects directly to the orthogonal transition state for ring-closure 9aTS. The decoupling of rotational and pseudopericyclic bond-forming transition states is contrasted with the single pericyclic transition state (15TS) for the electrocyclic ring-opening of oxetene (15) to acrolein (16). For comparison, the calculated homolytic strength of the N--NO bond is 40.0 kcal/mol (MP4(SDQ, FC)/6-31G*//MP2/6-31G* + ZPE).     

Mechanical properties of comonomer-compositionally fractionated poly[(3-hydroxybutyrate)-co-(3-mercaptopropionate)] with low 3-mercaptopropionate unit content

The mechanical properties, such as Young's modulus, yield strength, and the elongation at breakage, were investigated for several sulfur-containing biopolymers P(3HB-co-3MP). A series of P(3HB-co-3MP) samples with 3MP unit content ranging from 6.6 to 39.1 mol-% was biosynthesized by fermentation using the PHA-synthesizing bacteria Cupriavidus necator. For comparison, the bacterially synthesized P(3HB) and P(3HB-co-3HP) with the 3HP unit content ranging from 13.1 to 21.1 mol-% were also investigated. It was found that the sulfur-containing P(3HB-co-3MP) is much more durable to stretching. Notably, P(3HB-co-3MP) with the 3MP unit content of only 6.6 mol-% was found to show excellent mechanical properties.     

Gaussian-4 theory using reduced order perturbation theory

Two modifications of Gaussian-4 (G4) theory [L. A. Curtiss et al., J. Chem. Phys. 126, 084108 (2007)] are presented in which second- and third-order perturbation theories are used in place of fourth-order perturbation theory. These two new methods are referred to as G4(MP2) and G4(MP3), respectively. Both methods have been assessed on the G3/05 test set of accurate experimental data. The average absolute deviation from experiment for the 454 energies in this test set is 1.04 kcalmol for G4(MP2) theory and 1.03 kcalmol for G4(MP3) theory compared to 0.83 kcalmol for G4 theory. G4(MP2) is slightly more accurate for enthalpies of formation than G4(MP3) (0.99 versus 1.04 kcalmol), while G4(MP3) is more accurate for ionization potentials and electron affinities. Overall, the G4(MP2) method provides an accurate and economical method for thermochemical predictions. It has an overall accuracy for the G3/05 test set that is much better than G3(MP2) theory (1.04 versus 1.39 kcalmol) and even better than G3 theory (1.04 versus 1.13 kcalmol). In addition, G4(MP2) does better for challenging hypervalent systems such as H(2)SO(4) and for nonhydrogen species than G3(MP2) theory.