Novel real-time sensors to quantitatively assess in vivo inositol 1,4,5-trisphosphate production in intact cells

Real-time observation of messenger molecules in individual intact cells is essential for physiological studies of signaling mechanisms. We have developed a novel inositol 1,4,5-trisphosphate (IP(3)) sensor based on the pleckstrin homology (PH) domain from phospholipase C (PLC) delta. The environmentally sensitive fluorophore 6-bromoacetyl-2-dimethyl-aminonaphtalene was conjugated to the genetically introduced cysteine at the mouth of the IP(3) binding pocket for enhanced IP(3) selectivity and for rapid and direct visualization of intracellular IP(3) > or = 0.5 microM as fluorescence emission decreased. The probe, tagged with arginine-rich sequences for efficient translocation into various cell types, revealed a major contribution of Ca2+ influx to PLC-mediated IP(3) production that boosts Ca2+ release from endoplasmic reticulum. Thus, our IP(3) probe was extremely effective to quantitatively assess real-time physiological IP(3) production via those pathways formed only in the intact cellular configuration.     

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).     

Functional reassembly of a split PH domain

The pleckstrin homology (PH) domain forms a structurally conserved protein module of approximately 120 amino acid residues. Several proteins involved in cellular signaling and cytoskeletal organization possess split PH domains while their biological roles and ligand binding activity remain to be clarified. We have designed a split PH domain from a structurally well-characterized PH domain of phospholipase Cdelta(1) by dissecting the PH domain and tethering a coiled coil module to each subunit to ask a question of whether the coiled coil could mediate a functional reassembly of the split PH domain. Isothermal titration microcalorimetry measurements indicated a formation of a thermodynamically stable 1:1 complex of the N-terminal and C-terminal halves of the split PH domain by the coiled coil formation. The reassembled split PH domain binds to IP(3), a target molecule of the parent PLCdelta(1) PH domain, but not to L-IP(3), indicating that the split PH domain maintains a binding selectivity similar to the native PLCdelta(1) PH domain. These results demonstrate that the split PH domain folds into a functional structure when the split halves are brought to close proximity, and suggest that the native split PH domains, such as found in PLCgamma(1), have distinctive functions upon the reassembly.     

Design and synthesis of biotinylated inositol phosphates relevant to the biotin-avidin techniques

Six bifunctional molecules containing biotin and various inositol phosphates were synthesized. These compounds were designed on the basis of X-ray structures of the complexes of D-myo-inositol 1,4,5-triphosphates (IP(3)) and phospholipase C delta pleckstrin homology domain (PLCdelta PH) considering the application to the biotin-avidin techniques. The building blocks of the inositol moiety were synthesized starting with optically resolved myo-inositol derivatives and assembled to the biotin linker through a phosphate linkage.     

Accurate prediction of the bound form of the Akt pleckstrin homology domain using normal mode analysis to explore structural flexibility

Molecular docking is often performed with rigid receptors. This can be a serious limitation, since the receptor often differs between bound and unbound forms or between bound forms with different ligands. We recently developed a normal-mode based docking method and showed that it is possible to obtain reasonable estimates of the complexed form of the pleckstrin homology (PH) domain of Akt, starting with the free form of the receptor. With inositol (1,3,4,5)-tetrakisphosphate (IP4) as the ligand the docked results agree with the known high-resolution X-ray crystal structure of the IP4-Akt PH domain complex. We also tested our methods with PH4, SC66, and PIT-1, several recently designed PH domain inhibitors. The results are shown to be consistent with available experimental data and previous modeling studies. The method we described can be used for molecular docking analysis even when only an approximation of the experimental structure or model is known.     

Synthesis and properties of dendrimers possessing the same fluorophore(s) located either peripherally or off-center

Two series of phosphorus dendrimers functionalized by maleimide derivatives are synthesized, as well as three new monomeric maleimide derivatives, of which two are characterized by X-ray diffraction. The first series of phosphorus dendrimers possesses maleimide derivatives as end groups (6-48, from generation 0 to generation 3). The second series of dendrimers possesses a single copy of the same maleimide derivative linked "off-center" to a cyclotriphosphazene core, leading to dissymmetrical dendrimers; this series is synthesized from generation 0 to generation 2. The fluorescence properties of both series of dendrimers and of monomers are studied, affording new information. First, the presence of labile hydrogen extinguishes the fluorescence. Second, the grafting of the fluorophore(s) directly to the core affords highly fluorescent compounds. Finally, an original influence of the branches possessing phosphorhydrazone linkages toward the fluorescence properties is shown.     

A near IR di-styryl BODIPY-based ratiometric fluorescent chemosensor for Hg(II)

A novel BODIPY-based near-IR emitting probe as a selective and sensitive fluorophore for Hg(II) is synthesized. This versatile BODIPY fluorophore is functionalized for long wavelength emission at the 3 and 5 positions via a condensation reaction in which two dithiodioxomonoaza-based crown-containing phenyl units are conjugated to the BODIPY core as a chelating unit. This designed fluorophore, employing an ICT sensor can be used effectively to detect Hg(II) cations by way of a hypsochromic shift (∼90 nm) in both the absorption and emission spectra.     

A thermal dehydrogenative diels-alder reaction of styrenes for the concise synthesis of functionalized naphthalenes

Functionalized naphthalenes are valuable building blocks in many important areas. A microwave-assisted, intramolecular dehydrogenative Diels-Alder reaction of styrenyl derivatives to provide cyclopenta[b]naphthalene substructures not previously accessible using existing synthetic methods is described. The synthetic utility of these uniquely functionalized naphthalenes was demonstrated by a single-step conversion of one of these cycloadducts to a fluorophore bearing a structural resemblance to Prodan.     

Synthesis of New Functionalized Organophosphorus Acids and their Derivatives

Abstract

Functionalized organophosphorus acids and their derivatives are of great interest as chelating ligands, bioactive substances with various properties and the key compounds for the synthesis of phosphorus containing peptides. We have developed the convenient methods of the synthesis of new functionalized organophosphorus acids and their derivatives using a series of PH-acids and their esters with highly reactive fragments PH and POSi as well as phosphorus containing arnines with fragments PCHNH and PCHNSi. These organophosphorus synthons react smoothly with α-heterosubstituted derivatives of various amines and amides, functionalized alkenes and their oxides, as well as with acyl and sulfonyl chlorides giving the perspective compounds with high yields. New available types of new functionalized organophosphorus acids and their derivatives are presented below.     

Synthesis and Reactivity of the New Trimethylsilyl Esters of Aminomethylenebisorganophosphorus Acids

The interaction of trimethylsilyl esters of trivalent organophosphorus acids containing PH and POSiMe₃ fragments in the presence of trimethylsilyl trifluoromethanesulfonate as catalyst is proposed as a convenient method for the synthesis of new trimethylsilyl esters of aminomethylenebisorganophosphorus acids with three and four coordinated phosphorus. Also the new functionalized derivatives of the aminomethylenebisphosphinic acids with substituted hydroxymethyl moieties are synthesized, and some properties of the obtained compounds are presented.     

Fluorescent dendritic polymers and nanostructured coatings for the detection of chemical warfare agents and other analytes

Polyamidoamine (PAMAM) dendrimers of generations zero (G0) to four (G4), and a hyperbranched polyurea (HB‐PU), were functionalized with 1,5‐dansyl (1,5‐D), 2,5‐dansyl (2,5‐D), 2,6‐dansyl (2,6‐D) and nitrobenzofurazan (NBD) fluorophores that change their fluorescence emission wavelength in response to chemical environment, and the resulting dendritic polymers were characterized by MALDI‐TOF mass spectrometry, ¹H NMR, ¹³C NMR, and fluorescence spectroscopy. Fluorophore‐functionalized dendritic polymers were then reacted further with 3‐acryloxypropyldimethoxymethylsilane (AOP‐DMOMS) at various fluorophore to DMOMS substitution ratios. The resulting materials were cast onto glass slides, and cured into robust nanostructured coatings. Coatings with 50% fluorophore–50% DMOMS substitution showed the strongest fluorescence and the best physical properties. Coated coupons were tested against a wide range of analytes including the chemical warfare agent simulants dimethyl methylphosphonate (DMMP) and chloroethylethylsulfide (CEES), and the water‐methanol‐ethanol series. It was found that the ability of the coatings to distinguish between analytes decreased with increasing cross‐link density for both dendrimer and hyperbranched polymer‐based coatings. It was also found that the percent fluorophore substitution and the type of dendritic polymer carrying the fluorophore had no significant effect upon fluorescence emission wavelength, but fluorescence emission wavelength became less dependent upon solvent with increasing dendrimer generation and molecular mass. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 5101–5115, 2009     

The Role of Functional Moieties on Carbon Nanotube Surfaces in Solar Energy Conversion

An easily dispersible multiwalled carbon nanotube (MWCNT) derivative is prepared, and provides a platform for the synthesis of the phenyl butyric acid methyl ester (PCBM) analog. The carbene addition reaction of MWCNTs makes derivatives that are less soluble in organic solvents; by exploiting this differential solubility, PCBM analogs can be separated from the unreacted functionalized MWCNTs. Our experimental evidences indicate that it is the unique properties of the butyric acid methyl ester moiety that makes the acceptor material perform better in organic photovoltaics (OPVs). Studying the combination of the butyric acid methyl ester moiety and the deagglomerated functionalized MWCNT structures provides us an insight into nanoscale charge transfer and transportation inside the donor–acceptor domain. It is demonstrated that a strong structure–property relationship exists for the functionalized MWCNTs, which enables us to correlate the functionality on the carbon nanostructures with performance in OPVs.     

Synthesis and characterization of fluorescent cobalamin (CobalaFluor) derivatives for imaging

Fluorescent derivatives of cobalamin have been prepared by linking fluorophores to cobalamin through a propylamide spacer. Fluorescein, naphthofluorescein, and Oregon Green derivatives have been prepared in good yield by reaction of the fluorophore NHS-ester with beta-(3-aminopropyl)cobalamin to form fluorescent cobalamin conjugates (CobalaFluors) that are potentially suitable for the in vitro and in vivo imaging of transcobalamin receptors on cancer cells.     

Pleckstrin homology domain of phospholipase C-gamma1 directly binds to 68-kDa neurofilament light chain

Phosphoinositide-specific phospholipase C-gamma1 (PLC-gamma1) has two pleckstrin homology (PH) domains: an amino-terminal domain (PH1) and a split PH domain (PH2). Here, we show that overlay assay of bovine brain tubulin pool with glutathione-S-transferase (GST)-PLC-gamma1 PH domain fusion proteins, followed by matrix-assisted laser-desorption ionization-time of flight mass spectrometry (MALDI-TOF MS), identified 68-kDa neurofilament light chain (NF-L) as a binding protein of amino-terminal PH domain of PLC-gamma1. NF-L is known as a component of neuronal intermediate filaments, which are responsible for supporting the structure of myelinated axons in neuron. PLC-gamma1 and NF-L colocalized in the neurite in PC12 cells upon nerve growth factor stimulation. In vitro binding assay and immunoprecipitation analysis also showed a specific interaction of both proteins in differentiated PC12 cells. The phosphatidylinositol 4, 5-bisphosphate [PI(4,5)P(2)] hydrolyzing activity of PLC-gamma1 was slightly decreased in the presence of purified NF-L in vitro, suggesting that NF-L inhibits PLC-gamma1. Our results suggest that PLC-gamma1-associated NF-L sequesters the phospholipid from the PH domain of PLC-gamma1.     

Metal-enhanced fluorescence of nano-core–shell structure used for sensitive detection of prion protein with a dual-aptamer strategy

Metal-enhanced fluorescence (MEF) as a newly recognized technology is widespread throughout biological research. The use of fluorophore–metal interactions is recognized to be able to alleviate some of fluorophore photophysical constraints, favorably increase both the fluorophore emission intensity and photostability. In this contribution, we developed a novel metal-enhanced fluorescence (MEF) and dual-aptamer-based strategy to achieve the prion detection in solution and intracellular protein imaging simultaneously, which shows high promise for nanostructure-based biosensing. In the presence of prion protein, core–shell Ag@SiO₂, which are functionalized covalently by single stranded aptamer (Apt1) of prions and Cyanine 3 (Cy3) decorated the other aptamer (Apt2) were coupled together by the specific interaction between prions and the anti-prion aptamers in solution. By adjusting shell thickness of the pariticles, a dual-aptamer strategy combined MEF can be realized by the excitation and/or emission rates of Cy3. It was found that the enhanced fluorescence intensities followed a linear relationship in the range of 0.05–0.30 nM, which is successfully applied to the detection of PrP in mice brain homogenates.     

Synthesis and reactivity of substituted alkoxymethylphosphonites and their derivatives

Alkoxy‐substituted methylphosphonites and their derivatives are prepared using an organomagnesium method of synthesizing the organophosphorus compounds and alkoxymethylation of various PH acids and their derivatives. Also, certain properties of these promising compounds as important precursors of new functionalized organophosphorue compounds with alkoxymethyl fragments are presented. © 2012 Wiley Periodicals, Inc. Heteroatom Chem 23:281–289, 2012; View this article online at wileyonlinelibrary.com . DOI 10.1002/hc.21015     

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.     

Dendritic cell internalization of foam-structured fluorescent mesoporous silica nanoparticles

In this paper, foam-structured fluorescent mesoporous silica nanoparticles (FMSNs) are produced in a sol-gel method with the introduction of a phosphonate functional group. It is found that the phosphonate functionalized FMSNs with the foam structure minimizes the aggregation of FMSNs in solution. The average particle size of the FMSNs without and with phosphonate functionalization is 46.3 ± 5 nm and 60.5 ± 8 nm in diameter, respectively. The latter one exhibits higher fluorophore loading capacity (~67 ± 2.5%). The excitation wavelength (λ(ex)) of FMSNs is observed at 526 nm, approximate 12 nm larger in the Stoke-shift compared to the free organic dye at 494/514 nm. Furthermore, the photostability of the hydrophobic fluorophore is greatly improved by the FMSNs with the foam structure. In addition, the dose-dependent nature of FMSN uptake is assessed for the immune cells, the bone marrow-derived dendritic immune cells (BMDCs). Our results indicate that approximately 42% of BMDCs are able to take up foam-structured FMSNs (>5 μg/ml) without decreasing the viability of BMDCs. Thus, the phosphonate functionalized FMSNs with the foam structure are suitable to be used for many biomedical applications, especially in cell tracking.     

Screening search for organic fluorophores: syntheses and fluorescence properties of 3-azolyl-7-diethylaminocoumarin derivatives

In order to find a highly sensitive fluorophore, 3-azolyl-7-diethylaminocoumarin derivatives were synthesized. Both the absorption and fluorescence maxima of the coumarin-thiazole compounds showed red shifts with increases of the molar absorptivities and fluorescence intensities, in comparison with those of the corresponding coumarin-oxazole compounds. Among them, 3-(5-ethoxycarbonyl-1,3-thiazol-2-yl)-7-diethylamino-2H-chromen -2-one (3e) was one of the most promising candidates as a fluorophore accessible for analytical purposes in the fields of analytical and biological chemistry.     

Migratory insertion in N-heterocyclic carbene-containing Fe carbonyl complexes: an experimental and theoretical study

The compound [Fe(eta-C5H5)(CO)2(Me)] reacts thermally with N-heterocyclic carbenes (L) to give both alkyl, [Fe(eta-C5H5)(L)(CO)(Me)], and acyl, [Fe(eta-C5H5)(L)(CO)(COMe)], derivatives. The reaction temperature has been shown to affect the product distribution. The alkyl and acyl derivatives exist in an equilibrium that is more easily perturbed than in the tertiary phosphine analogues. DFT studies on the reactivity of [Fe(eta-C5H5)(CO)2(Me)] with PH3 and dihydroimidazole-2-ylidene (IH) have shown that CO exchange is energetically favoured for IH, and energetically disfavoured for PH3. The products of CO-induced migratory insertion, [Fe(eta-C5H5)(L)(CO)(COMe)], are more stable than the parent alkyl, [Fe(eta-C5H5)(L)(CO)(Me)], compounds. This stabilisation is larger when L = IH than when L = PH3. Stabilisation of the transition state by agostic interactions was seen in both instances, but this was significantly more pronounced for L = IH.