Biology-enabling inositol phosphates, phosphatidylinositol phosphates and derivatives

This review highlights inositol polyphosphate- and phosphatidylinositol-based small molecule probes that have advanced our understanding of intracellular signalling.     

Probing the interaction of the biotin-avidin complex with the relaxivity of biotinylated Gd-DTPA

A biotinylated Gd-DTPA complex is designed to study the biotin-avidin complexation using the longitudinal relaxivity of this new MRI label, which illustrates the use of MRI contrast agents to probe the formation of supramolecular assemblies in water.     

Design and synthesis of a potent biotinylated Smac mimetic

A biotinylated Smac mimetic (2) was designed based upon our previously reported potent, conformationally constrained Smac mimetic (1). Smac mimetic (2) was synthesized and determined to bind to XIAP protein with a high-affinity (K_i value of 13 nM) and is therefore a useful pharmacological tool for probing the intracellular targets of this class of potent Smac mimetics.     

Comparison by QCM and photometric enzymatic test of the biotin-avidin recognition on a biotinylated polypyrrole

By gravimetric measurements using a quartz cristal microbalance (QCM), we have studied the immobilization of biotinylated glucose oxidase enzymes (B-GOx) bound through on an intermediate avidin layer to a biotinylated polypyrrole film. The aim is to assess the amount of B-GOx specifically anchored on the biotinylated polypyrrole/avidin assembly thank to the biotin/avidin interaction between avidin and B-GOx. Indeed the estimated amount from the QCM measurement corresponds to the specific recognition of avidin/B-GOx added to a non-specific recognition (adsorption) of B-GOx. In order to discriminate these two phenomena, we have carried out a study by QCM of the anchoring of B-GOx on an avidin layer linked by adsorption to a polypyrrole free from biotin units. From QCM measurements we have deduced for the biotinylated polypyrrole/avidin assembly that the amount of B-GOx bound via the biotin/avidin interaction and those due to the avidin adsorption process correspond to 3.9 pmol cm(-2) (1.3 equivalent of B-Gox monolayer) and 1.4 pmol cm(-2) (0.46 equivalent of B-GOx monolayer) respectively. These values have been corroborated by measurements of the enzymatic activity of GOx.     

Design and synthesis of biotinylated dimethylation of alkannin oxime derivatives

DMAKO-05, a novel dimethylation of alkannin oxime derivative, exhibits remarkable anticancer activity as well as excellent cellular selectivity and thus has been considered as a promising antineoplastic agent for colorectal carcinoma and melanoma. However, its potent cytotoxicity is not closely associated with reactive oxygen species (ROS) and bioreductive alkylation. Its specific antitumor target(s) has still remained elusive. To recognize the molecular target(s) of DMAKO-05 and its analogs, four biotinylated DMAKO derivatives were designed and prepared. The biotin moiety was successfully introduced in the molecule through a modified Mitsunobu reaction, which kept its anticancer activity. Moreover, the cellbased investigation demonstrated that replacement of the linker C₄ chain with another alkyl chain (C₆ or C₈) gave rise to the enhancement of cytotoxicity. Among these biotinyl derivatives, both compound 16 and 8c exhibited more potent anticancer activity than DMAKO-05 against MCF-7 cells and were comparatively effective to alkannin toward HCT-15 cells. As expected, they might be thought as ideal chemical probes. Collectively, our present work could provide an available approach for the identification of the potential antineoplastic target(s) of DMAKO derivatives.     

Separation of fluorescent phosphatidyl inositol phosphates by CE

Phosphatidyl inositol 4,5-bisphosphate (PIP2) and phosphatidyl inositol 3,4,5-trisphosphate (PIP3) labeled with 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-propionic acid (BODIPY FL) on the acyl chain or a phosphatidyl ethanolamine head group were separated by CE with LIF detection. Several methods and capillary-coating procedures were tested for the separation of these phosphatidyl inositol phosphates (PIPs) at 20 degrees C. Separation of the PIPs in less than 20 min with excellent resolution was achieved using a buffer containing sodium deoxycholate (SDC), 1-propanol, MgCl2 and the polymer coating reagent, EOTrol LR. The efficiency of the optimized method was as high as 1.3x10(5) plates. The dependence of the separation on the concentration of 1-propanol, SDC, and MgCl2 was determined. The separation of PIP2 and PIP3 was primarily due to differential binding of the lipids to Mg2+ rather than to different solubilities in the micellar phase. The role of the SDC was to prevent adsorption of the hydrophobic lipids to the capillary wall and thus enhance the efficiency. The fluorescent PIPs are of value for both in vitro and in vivo assays of phospholipid metabolism. In particular, the use of these lipids with the optimized capillary-based separation will be of utility for drug screening as well as cell-based assays.     

Determination of inositol phosphates and other anions in rat brain

Anion chromatography methods have been developed to determine the concentrations of inositol phosphates and other water-soluble metabolites in rat brain. The cerebral cortex, hippocampus, and striatum were analyzed by chemically suppressed conductivity detection. Animals were sacrificed by directed microwave irradiation for more accurate estimations of in vivo concentrations. The effects of different sample preparation methods are compared. Trichloroacetic acid, formic acid, and perchloric acid extraction result in lower recoveries of phosphocreatine and other anions than does homogenization with water followed by chloroform-methanol extraction. The effects of postdecapitative ischemia on metabolite concentrations are determined. These methods will be used to study the effects of pharmacological agents on inositol phosphates and should be broadly applicable to the analysis of a variety of biological systems.     

Scale-up potential of ion-pair high-performance liquid chromatography method to produce biologically active inositol phosphates

This study was undertaken to evaluate the possibility that an analytical ion-pair HPLC procedure used to determine phytic acid (IP6) and its degradation products (IP3-IP5) can be transformed to a preparative purification method. A commercial phytic acid (CPA) preparation was separated into its component fractions of IP3, IP4, IPS, and IP6 on two C18 columns (1.8 and 4.2 ml) using 51% methanol containing 0.6-1% tetrabutylammonium hydroxide as ion-pair reagent and 0-0.025 M formic acid (pH 4.3) as eluent at 1.7 and 3.0 cm/min linear velocity, respectively, and 40 degrees C. Elution was monitored at 40 degrees C by a refractive index detector. Reproducible separation of CPA into four well-resolved peaks on these columns was possible after optimizing method variables, particularly the concentration of ion-pair reagent in the injected sample (>1.5%). The same separations were obtained after CPA loads were scaled up 25 times on a steel column (15 cm x 19 mm I.D.), packed with Ethyl C2 sorbent (10 microm) and on a 25 cm x 21.2 mm I.D. C18 column, but at a reduced linear velocity to increase the resolution. Therefore, this optimization of separation not only is useful for analysis of phytic acid and its degradation products but also it provides key parameters for scale up for further fractionation and characterization.     

Facile solid-phase synthesis of biotinylated alkyl thiols

Biotinylated alkyl thiols with the capacity to graft avidin proteins are in increasing demand for the development of self-assembled monolayers on gold. Here we propose 2-Chlorotrityl Chloride solid-phase resin as a new platform to produce these functionalized alkyl thiols. Biotinylated alkyl thiols of non-obvious solution synthesis were obtained rapidly using this method and without previous purification steps.     

Nonaqueous oxidation of phosphites to phosphates in nucleotide synthesis

$\text{m}$-Chloroperbenzoic acid in methylene chloride represents a rapid non-aqueous method for the conversion of phosphites to phosphates during oligonucleotide synthesis both in solution and on a solid support.     

Preparation of samples for high-performance liquid chromatography of inositol phosphates.

A simple method is described for the removal of extraneous material from tissue extracts prior to anion-exchange high-performance liquid chromatography of inositol phosphates. Samples are prepared by extraction with trichloroacetic acid or perchloric acid followed by removal of the excess acid. The extracts are then passed through small Dowex-50 cation-exchange columns and eluted with water. Dowex-50 pretreatment removes most of the ultraviolet absorbing material and cations from the samples but does not alter the content of inositol phosphates. This treatment results in improved reliability of chromatography, especially with respect to weakly retained molecules such as adenosine 5'-phosphate and the isomers of inositol monophosphate. In addition, sample pretreatment improves the useful lifetime of the analytical anion-exchange columns.     

Design and synthesis of an inositol phosphate analog based on computational docking studies

A virtual library of 54 inositol analog mimics of In(1,4,5)P₃ has been docked, scored, and ranked within the binding site of human inositol 1,4,5-trisphosphate 3-kinase A (IP₃-3KA). Chemical synthesis of the best scoring structure that also met distance criteria for 3′-OH to -P in phosphate has been attempted along with the synthesis of (1S,2R,3S,4S)-3-fluoro-2,4-dihydroxycyclohexanecarboxylic acid as an inositol analog, useful for non-invasive visualization and quantitation of IP₃-3KA enzymatic activity.     

Biomimetic one-pot synthesis of nucleotide phosphates

A strongly hydrophobic rigid diammonium, 1, an efficient extracting and transporting phase transfer reagent with high specificity for the pyrophosphate grouping, was used to synthesize ADP, ATP or ADP-NH₂ in a hydrophobic medium. Thus, practically pure ADP-NH₂ was obtained in 65% yield within 2 min.     

Electrochemical synthesis of substituted trialkyl phosphates

The process of synthesis of tryalkyl phosphates, i.e., derivatives of methanol, butanol, and diethylene glycol, by anodic oxidation of dimethyl phosphite in the corresponding alcohol is studied. It is found that three alkyl phosphates corresponding to the three possible combinations of substituents bound to phosphorus in phosphate are formed. It is established that the cause for formation of two of the three oxidation products is reesterification of the initial dimethyl phosphite by the alcohol in the course of electrolysis. It is assumed that the process of reesterification is catalyzed by the acid formed in the anode layer.     

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.     

HPLC with inductively coupled plasma optical emission spectrometric detection for the analysis of inositol phosphates

The use of inductively coupled plasma optimal emission spectroscopy as a detector for the high-performance liquid chromatographic analysis of inositol phosphates is studied. It is found that separation of different inositol phosphates with a mobile phase consisting of tetraethylammonium (0.14%, w/v), methanol (5%, v/v), and formic acid (0.18%, w/v) may be obtained on a PRP-1 column with an analysis time of 18 min. In addition, high specificity and sensitivity of the detection system used permits detection of the inositol phosphates from bi- to hexaphosphate free from interference of other chromatographic peaks, which could be from the sample or mobile phase. Additionally, it is possible to use less sample because of the high sensitivity of the detection system.     

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.