Simple synthesis of 32P-labelled inositol hexakisphosphates for study of phosphate transformations.

BACKGROUND AND AIMS: In many soils inositol hexakisphosphate in its various forms is as abundant as inorganic phosphate. The organismal and geochemical processes that exchange phosphate between inositol hexakisphosphate and other pools of soil phosphate are poorly defined, as are the organisms and enzymes involved. We rationalized that simple enzymic synthesis of inositol hexakisphosphate labeled with 32P would greatly enable study of transformation of soil inositol phosphates when combined with robust HPLC separations of different inositol phosphates. METHODS: We employed the enzyme inositol pentakisphosphate 2-kinase, IP5 2-K, to transfer phosphate from [γ-32P]ATP to axial hydroxyl(s) of myo-, neo- and 1D-chiro-inositol phosphate substrates. RESULTS: 32P-labeled inositol phosphates were separated by anion exchange HPLC with phosphate eluents. Additional HPLC methods were developed to allow facile separation of myo-, neo-, 1D-chiro- and scyllo-inositol hexakisphosphate on acid gradients. CONCLUSIONS: We developed enzymic approaches that allow the synthesis of labeled myo-inositol 1,[32P]2,3,4,5,6-hexakisphosphate; neo-inositol 1,[32P]2,3,4,[32P]5,6 - hexakisphosphate and 1D-chiro-inositol [32P]1,2,3,4,5,[32P]6-hexakisphosphate. Additionally, we describe HPLC separations of all inositol hexakisphosphates yet identified in soils, using a collection of soil inositol phosphates described in the seminal historic studies of Cosgrove, Tate and coworkers. Our study will enable others to perform radiotracer experiments to analyze fluxes of phosphate to/from inositol hexakisphosphates in different soils.

Insights into the activation mechanism of class I HDAC complexes by inositol phosphates.

Histone deacetylases (HDACs) 1, 2 and 3 form the catalytic subunit of several large transcriptional repression complexes. Unexpectedly, the enzymatic activity of HDACs in these complexes has been shown to be regulated by inositol phosphates, which bind in a pocket sandwiched between the HDAC and co-repressor proteins. However, the actual mechanism of activation remains poorly understood. Here we have elucidated the stereochemical requirements for binding and activation by inositol phosphates, demonstrating that activation requires three adjacent phosphate groups and that other positions on the inositol ring can tolerate bulky substituents. We also demonstrate that there is allosteric communication between the inositol-binding site and the active site. The crystal structure of the HDAC1:MTA1 complex bound to a novel peptide-based inhibitor and to inositol hexaphosphate suggests a molecular basis of substrate recognition, and an entropically driven allosteric mechanism of activation.

Synthetic inositol phosphate analogs reveal that PPIP5K2 has a surface-mounted substrate capture site that is a target for drug discovery.

Diphosphoinositol pentakisphosphate kinase 2 (PPIP5K2) is one of the mammalian PPIP5K isoforms responsible for synthesis of diphosphoinositol polyphosphates (inositol pyrophosphates; PP-InsPs), regulatory molecules that function at the interface of cell signaling and organismic homeostasis. The development of drugs that inhibit PPIP5K2 could have both experimental and therapeutic applications. Here, we describe a synthetic strategy for producing naturally occurring 5-PP-InsP4, as well as several inositol polyphosphate analogs, and we study their interactions with PPIP5K2 using biochemical and structural approaches. These experiments uncover an additional ligand-binding site on the surface of PPIP5K2, adjacent to the catalytic pocket. This site facilitates substrate capture from the bulk phase, prior to transfer into the catalytic pocket. In addition to demonstrating a "catch-and-pass" reaction mechanism in a small molecule kinase, we demonstrate that binding of our analogs to the substrate capture site inhibits PPIP5K2. This work suggests that the substrate-binding site offers new opportunities for targeted drug design.

Regioselective opening of myo-inositol orthoesters: mechanism and synthetic utility.

Acid hydrolysis of myo-inositol 1,3,5-orthoesters, apart from orthoformates, exclusively affords the corresponding 2-O-acyl myo-inositol products via a 1,2-bridged five-membered ring dioxolanylium ion intermediate observed by NMR spectroscopy. These C-2-substituted inositol derivatives provide valuable precursors for rapid and highly efficient routes to 2-O-acyl inositol 1,3,4,5,6-pentakisphosphates and myo-inositol 1,3,4,5,6-pentakisphosphate with biologically interesting and anticancer properties. Deuterium incorporation into the α-methylene group of such alkyl ester products (2-O-C(O)CD2R), when the analogous alkyl orthoester is treated with deuterated acid, is established utilizing the novel orthoester myo-inositol 1,3,5-orthobutyrate as an example. Such deuterated ester products provide intermediates for deuterium-labeled synthetic analogues. Investigation into this selective formation of 2-O-ester products and the deuterium incorporation is presented with proposed mechanisms from NMR experiments.

Inframolecular acid-base and coordination properties towards Na(+) and Mg(2+) of myo-inositol 1,3,4,5,6-pentakisphosphate: a structural approach to biologically relevant species.

The myo-inositol phosphates (InsPs) are specific signalling metabolites ubiquitous in eukaryotic cells. Although Ins(1,3,4,5,6)P(5) is the second most abundant member of the InsPs family, its certain biological roles are far from being elucidated, in part due to the large number of species formed by Ins(1,3,4,5,6)P(5) in the presence of metal ions. In light of this, we have strived in the past to make a complete and at the same time "biological-user-friendly" description of the Ins(1,3,4,5,6)P(5) chemistry with mono and multivalent cations. In this work we expand these studies focusing on the inframolecular aspects of its protonation equilibria and the microscopic details of its coordination behaviour towards biologically relevant metal ions. We present here a systematic study of the Ins(1,3,4,5,6)P(5) intrinsic acid-base processes, in a non-interacting medium, and over a wide pH range, analyzing the (31)P NMR curves by means of a model based on the Cluster Expansion Method. In addition, we have used a computational approach to analyse the energetic and structural features of the protonation and conformational changes of Ins(1,3,4,5,6)P(5), and how they are influenced by the presence of two physiologically relevant cations, Na(+) and Mg(2+).

The behaviour of inositol 1,3,4,5,6-pentakisphosphate in the presence of the major biological metal cations.

The inositol phosphates are ubiquitous metabolites in eukaryotes, of which the most abundant are inositol hexakisphosphate (InsP 6) and inositol 1,3,4,5,6-pentakisphosphate [Ins(1,3,4,5,6)P5)]. These two compounds, poorly understood functionally, have complicated complexation and solid formation behaviours with multivalent cations. For InsP 6, we have previously described this chemistry and its biological implications (Veiga et al. in J Inorg Biochem 100:1800, 2006; Torres et al. in J Inorg Biochem 99:828, 2005). We now cover similar ground for Ins(1,3,4,5,6)P5, describing its interactions in solution with Na+, K+, Mg2+, Ca2+, Cu2+, Fe2+ and Fe3+, and its solid-formation equilibria with Ca2+ and Mg2+. Ins(1,3,4,5,6)P5 forms soluble complexes of 1:1 stoichiometry with all multivalent cations studied. The affinity for Fe3+ is similar to that of InsP6 and inositol 1,2,3-trisphosphate, indicating that the 1,2,3-trisphosphate motif, which Ins(1,3,4,5,6)P5 lacks, is not absolutely necessary for high-affinity Fe3+ complexation by inositol phosphates, even if it is necessary for their prevention of the Fenton reaction. With excess Ca2+ and Mg2+, Ins(1,3,4,5,6)P5 also forms the polymetallic complexes [M4(H2L)] [where L is fully deprotonated Ins(1,3,4,5,6)P5]. However, unlike InsP6, Ins(1,3,4,5,6)P5 is predicted not to be fully associated with Mg2+ under simulated cytosolic/nuclear conditions. The neutral Mg2+ and Ca2+ complexes have significant windows of solubility, but they precipitate as [Mg4(H2L)] x 23H2O or [Ca4(H2L)] x 16H2O whenever they exceed 135 and 56 microM in concentration, respectively. Nonetheless, the low stability of the [M4(H2L)] complexes means that the 1:1 species contribute to the overall solubility of Ins(1,3,4,5,6)P 5 even under significant Mg2+ or Ca2+ excesses. We summarize the solubility behaviour of Ins(1,3,4,5,6)P5 in straightforward plots.

Regioselective hydrolysis of myo-inositol 1,3,5-orthobenzoate via a 1,2-bridged 2'-phenyl-1',3'-dioxolan-2'-ylium ion provides a rapid route to the anticancer agent Ins(1,3,4,5,6)P5.

Acid hydrolysis of myo-inositol 1,3,5-orthobenzoate leads regioselectively to 2-O-benzoyl-myo-inositol via a 1,2-bridged 2'-phenyl-1',3'-dioxolan-2'-ylium ion observed by 1H and 13C NMR spectroscopy, providing the precursor for a highly efficient route to the anticancer agent myo-inositol 1,3,4,5,6-pentakisphosphate.