Design and synthesis of the penta(acetoxymethyl) ester of dioctanoyl phosphatidylinositol-3,5-bisphosphate.

Extracellular administration of water-soluble and membrane-permeant analogs of phosphatidylinositol phosphates (PIPs) is a useful strategy for understanding the cellular roles of PIPs as well as the mode of action of drugs whose biological activity is associated with PIPs. We herein established the synthetic route to the dioctanoyl analogue of phosphatidylinositol 3,5-bisphosphate (di-C8-PI(3,5)P2) and its penta(acetoxymethyl) ester (di-C8-PI(3,5)P2/5AM).

Regioselective approach to phosphatidylinositol 3,5-bisphosphates: syntheses of the native phospholipid and biotinylated short-chain derivative.

A selective bis-silylation of 1D-O-TBDPS-myo-inositol leads to a 1,3,5-trisubstituted inositol, which can be advanced to the headgroup of phosphatidylinositol-3,5-bisphosphate [PI(3,5)P(2)]. A mild, regioselective method for construction of the diacylglycerol moiety containing differing fatty acid chains, including the naturally occurring lipids, was developed. Their union in the synthesis of the cell-signaling molecule PI(3,5)P(2) containing the sn-1-stearoyl and sn-2-arachidonoyl groups is described. The methodology was also used to generate dioctanoyl-PI(3,5)P(2) and a previously unreported biotin-PI(3,5)P(2) conjugate, which was coupled to neutravidin beads and used to pull down PI(3,5)P(2)-binding proteins from the cytosolic extract of adrenal neurosecretory cells. We report the specific pull-down of the PI(3,5)P(2)-binding protein svp1p, a known PI(3,5)P(2) effector involved in membrane trafficking.

Synthesis and biological evaluation of phosphatidylinositol phosphate affinity probes.

The synthesis of the complete family of phosphatidylinositol phosphate analogues (PIPs) from five key core intermediates A-E is described. These core compounds were obtained from myo-inositol orthoformate 1 via regioselective DIBAL-H and trimethylaluminium-mediated cleavages and a resolution-protection process using camphor acetals 10. Coupling of cores A-E with phosphoramidites 34 and 38, derived from the requisite protected lipid side chains, afforded the fully-protected PIPs. Removal of the remaining protecting groups was achieved via hydrogenolysis using palladium black or palladium hydroxide on carbon in the presence of sodium bicarbonate to afford the complete family of dipalmitoyl- and amino-PIP analogues 42, 45, 50, 51, 58, 59, 67, 68, 76, 77, 82, 83, 92, 93, 99 and 100. Investigations using affinity probes incorporating these compounds have identified novel proteins involved in the PI3K intracellular signalling network and have allowed a comprehensive proteomic analysis of phosphoinositide interacting proteins.

An unnatural PIP simulates growth factor signaling.

In this issue of Chemistry & Biology, Laketa et al. describe the synthesis of a membrane permeant phosphoinositide lipid that acts to stimulate PI(3,4,5)P(3)-dependent signaling without the need of growth factor stimulation.

Membrane-permeant phosphoinositide derivatives as modulators of growth factor signaling and neurite outgrowth.

Phosphoinositides are important signaling molecules that govern a large number of cellular processes such as proliferation, differentiation, membrane remodeling, and survival. Here we introduce a fully synthetic membrane-permeant derivative of a novel, easily accessible, and very potent phosphatidylinositol 3,4,5-trisphosphate [PtdIns(3,4,5)P(3)] mimic: phosphatidylinositol 3,4,5,6-tetrakisphosphate [PtdIns(3,4,5,6)P(4)]. The membrane-permeant PtdIns(3,4,5,6)P(4) derivative activated pathways downstream of phosphatidylinositol 3-kinase (PI3K), including protein kinase B, p70S6K, mitogen-activated protein kinase, and protein kinase C, more potently than similar membrane-permeant PtdIns(3,4,5)P(3) and PtdIns(3,4)P(2) derivatives in the absence of receptor stimulation. In addition, we demonstrate that treatment of PC12 cells with the membrane-permeant PtdIns(3,4)P(2), PtdIns(3,4,5)P(3), and PtdIns(3,4,5,6)P(4) derivatives increases the number of neurites per cell in the presence of NGF. This work establishes membrane-permeant phosphoinositides as powerful tools to study PI3K signaling and directly demonstrates that 3-phosphorylated phosphoinositides are instrumental for neurite initiation.

Synthesis of unsaturated phosphatidylinositol 4,5-bisphosphate and analogues.

A new approach for the synthesis of phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P2] is described, compatible with unsaturated fatty acid esters, as well as phosphorothioate and acetylenic analogues. This strategy depends on masking the phosphate charges with base-labile cyanoethyl esters, and the hydroxyls of the target with mild acid-labile protecting groups. A two-step basic then acidic global unblocking of orthogonal protecting groups provides the target lipid. A xanthenylidene acetal was used for key temporary protection of the 4,5-diol, and the 6-O was protected with a 1-(4-chlorophenyl)-4-ethoxypiperidin-4-yl (Cpep) acetal.

Biochemistry of inositol lipids.

Nature has created an immense combinatorial and structural heterogeneity among lipids. It is becoming increasingly accepted that the vast range of unique chemical entities encodes for distinct functions within biological systems. A unique group of lipids which stands out in terms of diversity as well as biological activity are inositol-containing lipids. The most well characterized inositol lipids are the phosphoinositides, phosphorylated derivatives of glycerophosphoinositol, which play a wide variety of cellular roles in many eukaryotic cells. Less well understood are ceramides containing inositol in fungi, and inositol glycolipids in pathogens. Here we review biochemical aspects of inositol-containing lipids with a focus on novel analytical procedures for their characterization.

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 molecular recognition of phosphatidylinositol-3-methylenephosphate.

[reaction: see text] Phosphatidylinositol-3-phosphate (PtdIns(3)P) is a spatial regulator of vesicular trafficking and other vital cellular processes. We describe the asymmetric total synthesis of a metabolically stabilized analogue, phosphatidylinositol-3-methylenephosphate (PtdIns(3)MP) from a differentially protected myo-inositol. NMR studies of PtdIns(3)MP bound to the (15)N-labeled FYVE domain showed significant (1)H and (15)N chemical shift changes relative to the unliganded protein.

Streamlined synthesis of phosphatidylinositol (PI), PI3P, PI3,5P2, and deoxygenated analogues as potential biological probes.

Highly direct total syntheses of phosphatidylinositol (PI), phosphatidylinositol-3-phosphate (PI3P), phosphatidylinositol-3,5-bisphosphate (PI3,5P2), and a range of deoxygenated versions are reported. Each synthesis is carried out to deliver the target in optically pure form. The key step for each synthesis is a catalytic asymmetric phosphorylation reaction that affects desymmetrization of an appropriate myo-inositol precursor. Elaboration to each target compound is then carried out employing a diversity-oriented strategy from the common precursors. In addition to three natural products, several additional streamlined total syntheses of deoxygenated PI analogues are reported. These syntheses set the stage for high-precision biological investigations of polar headgroup/biological target interactions of these membrane-associated signaling molecules.

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.

Asymmetric syntheses of phosphatidylinositol-3-phosphates with saturated and unsaturated side chains through catalytic asymmetric phosphorylation.

Highly direct asymmetric syntheses of phosphatidylinositol-3-phosphate (PI3P) in each enantiomerically pure form have been achieved. The key step involves catalytic asymmetric phosphorylation of meso-myo-inositol derivatives through desymmetrization. Protecting group schemes have been employed that allow for synthesis of PI3P with either saturated or arachidonate side chains, in analogy to the naturally occurring systems. Syntheses in each enantiomeric series are reported that rely on the choice of enantioselective peptide-based catalyst to define the enantiomeric series in which the syntheses are carried out.

3-Deoxy-3-substituted-D-myo-inositol imidazolyl ether lipid phosphates and carbonate as inhibitors of the phosphatidylinositol 3-kinase pathway and cancer cell growth.

3-Modified D-myo-inositol imidazolyl ether lipid phosphates and a carbonate were synthesized and evaluated as inhibitors of P13-K and Akt. These data are presented along with IC50 values for the inhibition of the growth of three cancer cell lines.

A versatile approach to PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3 from L-(-)-quebrachitol.

[reaction: see text] A versatile synthesis of PI(3,4)P2, PI(4,5)P2, and PI(3,4,5)P3 is disclosed, starting from L-(-)-quebrachitol, a byproduct of latex production. The crystalline nature of most intermediates and the utilization of inexpensive protecting groups facilitate this synthetic route and its scale-up.

The platelet cytoskeleton regulates the aggregation-dependent synthesis of phosphatidylinositol 3,4-bisphosphate induced by thrombin.

Pretreatment of intact platelets with cytochalasin D prevented actin polymerization and cytoskeleton reorganization induced by thrombin, but did not affect platelet aggregation. Under these conditions, synthesis of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) stimulated by thrombin was strongly inhibited, while production of phosphatidic acid was unaffected. The inhibitory effect of cytochalasin D was not observed when platelet aggregation was prevented by the RGDS peptide. We also found that cytochalasin D did not affect PtdIns(3,4)P2 synthesis induced by concanavalin A (ConA), which is known to occur through an aggregation-independent mechanism. Moreover, thrombin, but not ConA, induced the translocation of phosphatidylinositol 3-kinase to the cytoskeleton. This process was equally inhibited by both the RGDS peptide and cytochalasin D. These results demonstrate that the cytoskeleton represents a functional link between thrombin-induced aggregation and synthesis of PtdIns(3,4)P2.

A thiophosphate analog of dimyristoylphosphatidyl-inositol-4-phosphate is a substrate for mammalian phosphoinositide-specific phospholipase C.

1,2-Dimyristoyloxypropane-3-thiophosphate(rac-1-myo-inositol-4- phosphate), a thiophosphate analog of dimyristoyl phosphatidylinositol-4-phosphate was synthesized as a substrate for mammalian phosphoinositide-specific phospholipase C. Its activity with delta(1-132)-PI-PLC-delta 1 (a deletion mutant with the N-terminal pleckstrin homology domain removed) was studied in sonicated dispersions, with and without added Triton X-100. It had an initial activity of about 30 mumol min-1 mg-1, which rapidly decreased due to substrate depletion in the vesicle or micelle. The slower rate of hydrolysis appeared limited by enzyme hopping or exchange of substrate between vesicles or micelles, which was more rapid in the presence of detergent.

The headgroup orientation of dimyristoylphosphatidylinositol-4-phosphate in mixed lipid bilayers: a neutron diffraction study.

The trisodium salt of dimyristoylphosphatidylinositol-4-phosphate (DMPI-4P) has been synthesised specifically deuterated at particular sites in the headgroup. These materials have been used in neutron diffraction experiments, which successfully located the position (depth) of each of these deuterated sites to within +/- 0.5 A in a mixed model membrane (a 1:1 molar mixture of DMPI-4P with dimyristoyl-phosphatidylcholine, DMPC, in the L alpha phase, hydrated to the level of 28 water molecules per lipid molecule). The diffracted intensities were measured at four different D2O/H2O ratios and six orders of diffraction were obtained. These data sets, in conjunction with computer modelling, have been used to determine the orientation of the inositol ring of DMPI-4P, localising each vertical H-H distance to within approximately +/- 0.03 A. The orientation of the inositol ring is found to be one in which the C5 hydroxyl is extended out into the aqueous medium. This is, therefore, the most accessible site for water-borne reagents. This may be significant for the important pathway leading from PI-4P to PI-4,5P2. On the assumption that the P/ODAG bond is orientated parallel to the bilayer normal, these results are consistent with two possible conformations for the portion of the headgroup connecting the diacylglycerol to the inositol ring. Distinction between these two is difficult, but one may be favoured since the other involves close atom-atom contacts.