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 (IP6) occur, although for three of these (scyllo, flea, 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 n-chiro-IP6 and used them to identify signals from these compounds in three soils from the Falkland Islands. Both compounds resisted hypobromite oxidation and gave quantifiable P-31 NMR signals at delta = 6.67 ppm (equatorial phosphate groups of the 4-equatorial/2-axial conformer of neo-IP6) and delta = 6.48 ppm (equatorial phosphate groups of the 2-equatorial/4-axial conformer of D-chiro-IP6) 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% (n-chiro). Reappraisal of the literature based on the new signal assignments revealed that neo- and D-chiro-IP6 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-IP6 in the environment. The hypobromite oxidation and solution P-31 NMR spectroscopy procedure allows the simultaneous quantification of all four IP6 stereoisomers in environmental samples and provides a platform for research into the origins and ecological significance of these enigmatic compounds

Determination of <i>neo</i>- and d-<i>chiro</i>-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 (IP₆) occur, although for three of these (<i>scyllo</i>, <i>neo</i>, and d-<i>chiro</i>) the origins, dynamics, and biological function remain unknown, due in large part to analytical limitations in their measurement in environmental samples. We synthesized authentic <i>neo</i>- and d-<i>chiro</i>-IP₆ and used them to identify signals from these compounds in three soils from the Falkland Islands. Both compounds resisted hypobromite oxidation and gave quantifiable ³¹P NMR signals at δ = 6.67 ppm (equatorial phosphate groups of the 4-equatorial/2-axial conformer of <i>neo</i>-IP₆) and δ = 6.48 ppm (equatorial phosphate groups of the 2-equatorial/4-axial conformer of d-<i>chiro</i>-IP₆) in soil extracts. Inositol hexakisphosphate accounted for 46–54% of the soil organic phosphorus, of which the four stereoisomers constituted, on average, 55.9% (<i>myo</i>), 32.8% (<i>scyllo</i>), 6.1% (<i>neo</i>), and 5.2% (d-<i>chiro</i>). Reappraisal of the literature based on the new signal assignments revealed that <i>neo</i>- and d-<i>chiro</i>-IP₆ 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 <i>neo</i>- and d-<i>chiro</i>-IP₆ in the environment. The hypobromite oxidation and solution ³¹P NMR spectroscopy procedure allows the simultaneous quantification of all four IP₆ stereoisomers in environmental samples and provides a platform for research into the origins and ecological significance of these enigmatic compounds

Modulation of the substrate specificity of the kinase PDK1 by distinct conformations of the full-length protein

The activation of at least 23 different mammalian kinases requires the phosphorylation of their hydrophobic motifs by the kinase PDK1. A linker connects the phosphoinositide-binding PH domain to the catalytic domain, which contains a docking site for substrates called the PIF pocket. Here, we used a chemical biology approach to show that PDK1 existed in equilibrium between at least three distinct conformations with differing substrate specificities. The inositol polyphosphate derivative HYG8 bound to the PH domain and disrupted PDK1 dimerization by stabilizing a monomeric conformation in which the PH domain associated with the catalytic domain and the PIF pocket was accessible. In the absence of lipids, HYG8 potently inhibited the phosphorylation of Akt (also termed PKB) but did not affect the intrinsic activity of PDK1 or the phosphorylation of SGK, which requires docking to the PIF pocket. In contrast, the small-molecule valsartan bound to the PIF pocket and stabilized a second distinct monomeric conformation. Our study reveals dynamic conformations of full-length PDK1 in which the location of the linker and the PH domain relative to the catalytic domain determines the selective phosphorylation of PDK1 substrates. The study further suggests new approaches for the design of drugs to selectively modulate signaling downstream of PDK1