Metabolically Stabilized Derivatives of Phosphatidylinositol 4-Phosphate: Synthesis and Applications

Abstract

Summary Phosphatidylinositol-4-phosphate (PtdIns(4)P) is an essential component of eukaryotic membranes and a marker of the Golgi complex. The pivotal role of PtdIns(4)P in signaling and trafficking necessitates the development of synthetic derivatives of this lipid that are resistant to hydrolysis and therefore can be used in biochemical, structural and functional assays to monitor time-dependent processes at the Golgi membranes. Here, we developed novel metabolically stabilized (ms) analogues of the PtdIns(4)P lipid and the inositol 1,4-bisphosphate (IP2) head group derivative suitable for outfitting with a reporter tag and demonstrated that these compounds can substitute the natural lipid in peripheral protein recruitment and membrane deformation. The methylenephosphonate (MP) and phosphorothioate (PT) analogues of PtdIns(4)P and the aminohexyl (AH)-IP2 probe are recognized by the PtdIns(4)P-specific PH domain of four phosphate adaptor protein 1 (FAPP1). Binding of FAPP1 to the PtdIns(4)P derivatives stimulates insertion of the PH domain into the lipid layers and induces tubulation of membranes. Both ms-analogues and IP2-probes could be invaluable for identifying novel protein effectors and characterizing and monitoring PtdIns(4)P-dependent signaling cascades within the trans-Golgi network (TGN).

Development of isotope-enriched phosphatidylinositol-4- and 5-phosphate cellular mass spectrometry probes

“…In summary, we have developed robust, and efficient synthetic routes towards saturated and unsaturated PtdIns4P and PtdIns5P derivatives. To our knowledge, this work represents both the first synthesis of unsaturated PtdIns4P derivatives that relies on a desymetrisation strategy, [28][29][30] and the most efficient synthesis of enantiomerically pure PtdIns5P described so far, with an overall yield of 20%. 35,56,57 As a result of the high yielding enzymatic desymmetrisation strategy employed, these routes are amenable to the synthesis of a versatile set of stable-isotope labelled PtdInsP derivatives.…”

“…Regioselectivity of the reaction was further improved by using the bulkier TIPS group (Scheme S5). The 4-O-TIPS ether (−)-48 was thus obtained in 71% yield using TIPSOTf in the presence of 2,6-lutidine at −78 °C, and regioselectivity was confirmed by 1 H- 29 Si Heteronuclear Multiple Bond Correlation (HMBC) NMR analysis ( Figure S4).…”

“…These routes are therefore not applicable to the synthesis of expensive isotopeenriched materials. [28][29][30][31][32] Given the high cost of isotopically labelled materials, synthetic routes must be as concise as possible, scalable, and reproducible.…”

The Chemical Biology of Phosphoinositide 3‐Kinases

“…This was achieved by using phosphothioates and methylenephosphonates (for a very recent example of PtdIns(4)P (Scheme 3), see refs. [111,112], and references therein).…”

Molecular Analysis of Protein–Phosphoinositide Interactions

“…The PH domains of Grp1, Fapp1, and oxysterol binding protein recognize not only PIs but also membrane-attached Arf GTPases (Balla et al 2005; Cohen et al 2007; Godi et al 2004; Levine and Munro 2002). Recent biochemical and structural analysis of the Fapp1 PH domain reveals that the PtdIns(4)P and Arf1 binding sites are separate, which allow for the association with both ligands simultaneously and independently (He et al 2011a, b). This mode of ‘coincidence detection’ increases affinities and specificities of the PH domains toward membranes enriched in unique PIs and Arfs.…”