Chemical Synthesis and Molecular Recognition of Phosphatase-Resistant Analogues of Phosphatidylinositol-3-phosphate

Abstract

The remodeling of phosphatidylinositol polyphosphates in cellular membranes by phosphatases and kinases orchestrates the signaling by these lipids in space and time. In order to provide chemical tools to study of 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-monofluoromethylphosphonate 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 phosphorlyation reagent, monofluoromethylphosphonyl chloride, which has general applications for the preparation of "pKa-matched" monofluorophosphonates. These ms-PtdIns(3)P analogues exhibited reduced binding activities with 15 N-labelled FYVE and PX domains, as significant 1 H and 15 N chemical shift changes in the FYVE domain were induced by titrating ms-PtdIns(3)Ps into membrane-mimetic dodecylphosphocholine (DPC) micelles. In addition, the PtdIns(3)P analogues with dioleyl and dipalmitoyl chains were substrates for the 5-kinase enzyme PIKfyve; the corresponding phosphorylated ms-PI(3,5)P 2 products were detected by radio-TLC analysis.

PtdIns5P: News and views of its appearance, disappearance and deeds

“…Similar in vitro assays have also been conducted by an independent laboratory that used our mouse PIKfyve clone expressed by yeast plasmid [32]. Agreeably, our laboratory and that of Dr. Bob Michelle did not observe PtdIns5P when synthetic PtdIns3P substrate preparations were used in the in vitro assays, despite the great amounts of produced 32 P-PtdIns(3,5)P 2 [9,29,32] (illustrated in Fig. 2A).…”

“…PIKfyve was first implicated in PtdIns5P biosynthesis based on in vitro data from our laboratory, which revealed that native or recombinant PIKfyve preparations purified from various mammalian or insect cells synthesize PtdIns5P from PtdIns and PtdIns(3,5)P 2 , from PtdIns3P in the presence of [γ- 32 P]ATP [9,29–31]. Substrate side chains play a key role in the efficiency of the in vitro kinase assay, with naturally occurring acyl chains being greatly preferred over the synthetic di-C 16 [9].…”

“…2A). Lack of coproduced PtdIns5P along with PtdIns(3,5)P 2 has been observed using PtdIns3P substrates with either di-C 16 or di-C 18 side chains-from multiple vendors (Matreya; Echelon or Michelle’s lab) in in vitro assays conducted by different laboratories [9,29,32]. Thus, the experimental data in these studies had firmly demonstrated that PIKfyve-catalyzed PtdIns5P synthesis in vitro is strictly dependent on the presence of PtdIns substrates.…”

Lipid-anchored DNA mediates vesicle fusion as observed by lipid and content mixing

“…13,14 Here, 0.5 g of 1,2-O-dioctadecylrac-glycerol ͑0.83 mmol͒ and 0.32 mL diisopropylethylamine ͑1.8 mmol͒ were dissolved in 10 mL anhydrous dichloromethane at 0°C under a nitrogen atmosphere. Then, 0.25 g 2-cyanoethyl N, N-diisopropylchlorophosphoramidite ͑1.3 mmol͒ was added dropwise to the reaction, which was stirred at 0°C for 15 min, then at room temperature for 2 h. The crude product solution was washed with a 0.5 M solution of sodium bicarbonate, and the solvent evaporated.…”

Synthesis and Biological Activity of Metabolically Stabilized Cyclopentyl Trisphosphate Analogues of D‐myo‐Ins(1,4,5)P3

“…We have previously employed phosphorothioate and methylenephosphonates to prepare phosphatase-resistant analogues of phosphoinositides [8][9][10] and lysophosphatidic acid. [11] In each case, the analogues were less susceptible to acidic or enzymatic cleavage and some bound to cognate binding proteins with reduced affinity.…”