Synthesis of Modular Headgroup Conjugates Corresponding to All Seven Phosphatidylinositol Polyphosphate Isomers for Convenient Probe Generation

Abstract

Phosphatidylinositol polyphosphate lipids (PIPns) play key roles in important biological pathways, and defects in the signaling activities of these molecules have been implicated in a number of disease states. As such, it is necessary to understand the complex roles of these lipids in biological pathways, which often involve their actions as site‐specific ligands that recruit receptors to the surfaces of cellular membranes. However, the complex structures of PIPn family members, of which seven biologically active isomers exist, complicate studies. Derivatized analogs of the PIPn structures are beneficial as chemical tools for elucidating signaling and binding activities. Herein, we present an efficient approach to probe the generation in which amino conjugates of PIPn headgroups can be conveniently functionalized in the final step of the synthesis to obtain a number of derivatized analogs of use for different studies. In addition to the application of this strategy to generate PI‐(4,5)‐P2 headgroup probes, we also report the synthesis of PIPn–amine conjugates corresponding to all seven naturally existing isomers. This approach will be invaluable for generating the range of probe structures that is required to elucidate the intricacies of PIPn signaling. (© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2009)

Microarray analysis of Akt PH domain binding employing synthetic biotinylated analogs of all seven phosphoinositide headgroup isomers

“…(Dorman et al, 1995; Henne et al, 1988; Inoue et al, 1999; Marecek et al, 1992; Nakanishi et al, 2002; Olszewski et al, 1995; Prestwich et al, 1991; Schafer et al, 1990; Tegge and Ballou, 1992) Using this approach, we recently described the synthesis of amino-conjugates of all seven PIP n headgroup isomers ( 2a–g ), and efficient subsequent derivatization to produce different reporter-tagged analogs. (Gong et al, 2009a) In the synthesis of enantiomerically pure headgroup analogs, we followed previously devised procedures for myo -inositol desymmetrization that were developed by Bruzik and co-worker(Kubiak and Bruzik, 2003), and Holmes and co-workers(Conway et al, 2010; Painter et al, 1999). To synthesize ligands for binding analysis using all isomers, 2a–g were allowed to react with synthetic biotin–TEG–succinimidyl ester reagent 3 to generate PIP n –TEG–biotin conjugates 1a–g for studies.…”

“…Rabbit anti-GST antibody and Cy3 conjugated goat anti-rabbit IgG antibody were obtained from Novus Biologicals (Littleton, CO). Synthetic procedures for modular PIP n –amine conjugates 2a–g (Gong et al, 2009a), as well as biotin-TEG-succinimidyl ester 3 and biotinylated PI(4,5)P 2 analog 1c (Gong et al, 2009b) were previously described.…”

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

“…This probe can be further modified through the coupling to a fluorescent, biotinylated or photoaffinity tag, which is necessary for the detection and characterization of the PtdIns(4)P-related processes. The soluble aminohexyl (AH) IP 2 probe ( 23 ) was synthesized as shown in Figure 4 (Gong, et al, 2009). To protect the 4-position, intermediate 16 was first produced in four steps from myo -inositol using the camphor resolution protocol (Kubiak and Bruzik, 2003).…”

Global approaches for the elucidation of phosphoinositide-binding proteins

“…Here, PI(3,4,5)P 3 probe 7 was initially designed and synthesized, which contains both a benzophenone photoaffinity tag as well an alkynyl secondary label linked via a Y-shaped lysine linker. While this probe contains the PIP 3 headgroup, the glycerolipid backbone is simplified by using an aminohexyl chain to provide hydrophobicity and also allow for modular synthesis (Gong et al, 2009b). The alkynyl tag provides versatility in analysis, as a fluorescent dye can be clicked on for initial validation and optimization studies using gel electrophoresis, while alternatively a biotin moiety can be introduced via click chemistry for affinity enrichment of protein targets (Fig.…”