A dansyl-derivatized phytic acid analogue as a fluorescent substrate for phytases: experimental and computational approach.

A new myo-inositol pentakisphosphate was synthesized, which featured a dansyl group at position C-5. The fluorescent tag was removed from the inositol by a 6-atom spacer to prevent detrimental steric interactions in the catalytic site of phytases. The PEG linker was used in order to enhance hydrophilicity and biocompatibility of the new artificial substrate. Computational studies showed a favorable positioning in the catalytic site of phytases. Enzymatic assays demonstrated that the tethered myo-inositol was processed by two recombinant phytases Phy-A and Phy-C, classified respectively as acid and alkaline phytases, with similar rates of phosphate release compared to their natural substrate.

68 Ga-Ca-phytate particles: A potential lung perfusion agent of synthetic origin prepared in a cold kit format.

The objective of this study was to investigate the radiosynthesis of 68 Ga-Ca-phytate particles and then characterize the formulation for radiochemical purity, radioactive particle size distribution, and biodistribution in normal rats. This radiotracer was prepared using a commercial phytate cold kit after reconstitution with saline, 68 Ga-chloride generator eluent, calcium chloride, and air, then heating at 100°C for 30 minutes to achieve 99% radiochemical purity of 68 Ga-particles that were 21% 3-5 µm, 8% 5-15 µm, and 71% >15 µm in diameter. This optimal formulation was stable for 2 hours at room temperature. Intravenous administration of 68 Ga-particles in rats resulted in an uptake of 93% in the lungs, 4% in the liver plus spleen, and 3% in the carcass after 20 minutes. Two-thirds of the carcass activity was radioactive blood, likely to be 68 Ga-transferrin. The positron emission tomography image was superior than the 99m Tc-MAA image because it displayed high lung uptake against a low background. Low uptake by the liver, spleen did not interfere with the diagnostic quality, and faint activity in the submandibular (salivary) glands was due to 68 Ga-transferrin. The preclinical data so far indicate that 68 Ga-Ca-phytate particles have good potential as a lung perfusion imaging agent.

Synthesis of Biotinylated Inositol Hexakisphosphate To Study DNA Double-Strand Break Repair and Affinity Capture of IP6-Binding Proteins.

Inositol hexakisphosphate (IP6) is a soluble inositol polyphosphate, which is abundant in mammalian cells. Despite the participation of IP6 in critical cellular functions, few IP6-binding proteins have been characterized. We report on the synthesis, characterization, and application of biotin-labeled IP6 (IP6-biotin), which has biotin attached at position 2 of the myo-inositol ring via an aminohexyl linker. Like natural IP6, IP6-biotin stimulated DNA ligation by nonhomologous end joining (NHEJ) in vitro. The Ku protein is a required NHEJ factor that has been shown to bind IP6. We found that IP6-biotin could affinity capture Ku and other required NHEJ factors from human cell extracts, including the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), XRCC4, and XLF. Direct binding studies with recombinant proteins show that Ku is the only NHEJ factor with affinity for IP6-biotin. DNA-PKcs, XLF, and the XRCC4:ligase IV complex interact with Ku in cell extracts and likely interact indirectly with IP6-biotin. IP6-biotin was used to tether streptavidin to Ku, which inhibited NHEJ in vitro. These proof-of-concept experiments suggest that molecules like IP6-biotin might be used to molecularly target biologically important proteins that bind IP6. IP6-biotin affinity capture experiments show that numerous proteins specifically bind IP6-biotin, including casein kinase 2, which is known to bind IP6, and nucleolin. Protein binding to IP6-biotin is selective, as IP3, IP4, and IP5 did not compete for binding of proteins to IP6-biotin. Our results document IP6-biotin as a useful tool for investigating the role of IP6 in biological systems.

Design and synthesis of lipid-coupled inositol 1,2,3,4,5,6-hexakisphosphate derivatives exhibiting high-affinity binding for the HIV-1 MA domain.

The precursor of Gag protein (Pr55(Gag)) of human immunodeficiency virus, the principal structural component required for virus assembly, is known to bind d-myo-phosphatidylinositol 4,5-bisphosphate (PIP2). The N-terminus of Pr55(Gag), the MA domain, plays a critical role in the binding of Pr55(Gag) to the plasma membrane. Herein, we designed and synthesized myo-phosphatidylinositol 2,3,4,5,6-pentakisphosphate (PIP5) derivatives comprising highly phosphorylated inositol and variously modified diacylglycerol to examine the MA-binding properties. The inositol moiety was synthesized starting with myo-inositol and assembled with a hydrophobic glycerol moiety through a phosphate linkage. The Kd value for MA-binding of the PIP5 derivative 2 (Kd = 0.25 µM) was the lowest (i.e., highest affinity) of all derivatives, i.e., 70-fold lower than the Kd for the PIP2 derivative 1 (Kd = 16.9 µM) and 100-fold lower than the Kd for IP6 (Kd = 25.7 µM), suggesting the possibility that the PIP5 derivative blocks Pr55(Gag) membrane binding by competing with PIP2 in MA-binding.

Determination of neo- and D-chiro-inositol hexakisphosphate in soils by solution 31P NMR spectroscopy.

The inositol phosphates are an abundant but poorly understood group of organic phosphorus compounds found widely in the environment. Four stereoisomers of inositol hexakisphosphate (IP(6)) occur, although for three of these (scyllo, neo, and D-chiro) the origins, dynamics, and biological function remain unknown, due in large part to analytical limitations in their measurement in environmental samples. We synthesized authentic neo- and D-chiro-IP(6) and used them to identify signals from these compounds in three soils from the Falkland Islands. Both compounds resisted hypobromite oxidation and gave quantifiable (31)P NMR signals at δ = 6.67 ppm (equatorial phosphate groups of the 4-equatorial/2-axial conformer of neo-IP(6)) and δ = 6.48 ppm (equatorial phosphate groups of the 2-equatorial/4-axial conformer of D-chiro-IP(6)) in soil extracts. Inositol hexakisphosphate accounted for 46-54% of the soil organic phosphorus, of which the four stereoisomers constituted, on average, 55.9% (myo), 32.8% (scyllo), 6.1% (neo), and 5.2% (D-chiro). Reappraisal of the literature based on the new signal assignments revealed that neo- and D-chiro-IP(6) occur widely in both terrestrial and aquatic ecosystems. These results confirm that the inositol phosphates can constitute a considerable fraction of the organic phosphorus in soils and reveal the prevalence of neo- and D-chiro-IP(6) in the environment. The hypobromite oxidation and solution (31)P NMR spectroscopy procedure allows the simultaneous quantification of all four IP(6) stereoisomers in environmental samples and provides a platform for research into the origins and ecological significance of these enigmatic compounds.

Synthesis of an inositol hexakisphosphate (IP6) affinity probe to study the interactome from a colon cancer cell line.

Inositol hexakisphosphate (InsP6 or IP6) is an important signalling molecule in vesicular trafficking, neurotransmission, immune responses, regulation of protein kinases and phosphatases, activation of ion channels, antioxidant functions and anticancer activities. An IP6 probe was synthesised from myo-inositol via a derivatised analogue, which was immobilised through a terminal amino group onto Dynabeads. Systematic analysis of the IP6 interactome has been performed using the IP6 affinity probe using cytosolic extracts from the LIM1215 colonic carcinoma cell line. LC/MS/MS analysis identified 77 proteins or protein complexes that bind to IP6 specifically, including AP-2 complex proteins and ß-arrestins as well as a number of novel potential IP6 interacting proteins. Bioinformatic enrichment analysis of the IP6 interactome reinforced the concept that IP6 regulates a number of biological processes including cell cycle and division, signal transduction, intracellular protein transport, vesicle-mediated transport and RNA splicing.

Synthesis and nonradioactive micro-analysis of diphosphoinositol phosphates by HPLC with postcolumn complexometry.

A nonradioactive high-performance anion-exchange chromatographic method based on MDD-HPLC (Mayr Biochem. J. 254:585-591, 1988) was developed for the separation of inositol hexakisphosphate (InsP(6), phytic acid) and most isomers of pyrophosphorylated inositol phosphates, such as diphosphoinositol pentakisphosphate (PPInsP(5) or InsP(7)) and bis-diphosphoinositol tetrakisphosphate (bisPPInsP(4) or InsP(8)). With an acidic elution, the anion-exchange separation led to the resolution of four separable PPInsP(5) isomers (including pairs of enantiomers) into three peaks and of nine separable bisPPInsP(4) isomers into nine peaks. The whole separation procedure was completed within 20-36 min after optimization. Reference standards of all bisPPInsP(4) isomers were generated by a nonenzymatic shotgun synthesis from InsP(6). Hereby, the phosphorylation was brought about nonenzymatically when concentrated InsP(6) bound to the solid surface of anion-exchange beads was incubated with creatine phosphate under optimal pH conditions. From the mixture of pyrophosphorylated InsP(6) derivatives containing all theoretically possible isomers of PPInsP(5), bisPPInsP(4), and also some isomers of trisPPInsP(3), isomers were separated by anion-exchange chromatography and fractions served as reference standards of bisPPInsP(4) isomers for further investigation. Their isomeric nature could be partly assigned by comparison with position specifically synthesized or NMR-characterized purified protozoan reference compounds and partly by limited hydrolysis to PPInsP(5) isomers. By applying this nonradioactive analysis technique to cellular studies, the isomeric nature of the major bisPPInsP(4) in mammalian cells could be identified without the need to obtain sufficient material for NMR analysis.

Influence of phytic acid on zinc phosphate cement.

The influence of phytic acid on the properties of zinc phosphate cement was studied by adding 2-13 wt% phytic acid to the liquid. Improved mechanical strength and stability were found for some cements prepared from commercial powders when liquids with increased phytic acid content were used. The results indicate that the formation of increased amounts of zinc phytate has a favorable effect on the properties of zinc phosphate cement.

[99mTc]Ca-phytate: some colloidal characteristics related to the optimal preparation conditions.

Some physico-chemical characteristics of the colloidal radiopharmaceutical [99mTc]Ca-phytate related to optimal preparation conditions have been studied. It is demonstrated that the Ca2+-phytate stoichiometry is 6:1. Two different Ca-phytate colloids seem to be formed, mainly depending on the Ca2+:phytate molar ratio--one of low mycelar size for a 1:1 Ca2+:phytate molar ratio (cmc = 5.10(-5) M), and another one, with a higher mycelar size for a 6:1 molar ratio (cmc = 8.10(-5) M). This last one it probably better for providing a good quality splenic uptake.