Synthesis and Convenient Functionalization of Azide-Labeled Diacylglycerol Analogues for Modular Access to Biologically Active Lipid Probes

Abstract

Cell membrane lipids have been identified as key participants in cell signaling activities. One important role is their involvement as site-specific ligands in protein-membrane binding interactions, which result in the anchoring of peripheral proteins onto cellular membranes. These events generally regulate protein function and localization and have been implicated in both normal physiological processes and those pertaining to disease state onset. Thus, it is important to elucidate the details of interactions at the molecular level, such as lipid-binding specificities and affinities, the location of receptor binding domains and multivalency in binding. For this purpose, we have designed and developed azido-tagged lipid analogues as conveniently functionalizable lipid probe scaffolds. Herein, we report the design and synthesis of the initial structure of this type, diacylglycerol analogue 2, which contains an azide tag at the sn-1 position of the lipid headgroup. Direct functionalization of this compound with a range of reporter groups has been performed to illustrate the facile access to probes of use for characterizing binding. Quantitative lipid-binding studies using protein kinase C, a known DAG-binding receptor, demonstrate that these probes are active mimetics of natural DAG. Thus, these DAG probes will serve as robust sensors for studies aimed at understanding binding interactions and as precursors for the development of analogous probes of more complex phospholipids and glycolipids.

Modular synthesis of bis(monoacylglycero)phosphate for convenient access to analogues bearing hydrocarbon and perdeuterated acyl chains of varying length

“…In fact, quantitative sn-2-to-sn-3 migration has been reported in the synthesis of BMPs and other glycerolipids under certain conditions. 16,27 Based on this evidence, the reverse sn-3-to-sn-2 migration was not expected to be problematic in the deprotection of 9a-h. This was confirmed via 1 H NMR analysis, as the typical chemical shifts for the hydrogens on the (S,S)-3,3 0 -BMP isomer (wd3.9-d4.2) dominated the spectra of 10a-h (see 1 H NMR spectra in Supplementary data), with minimal of the characteristic peaks of the (S,S)-2,2 0 -isomer (wd 3.8, d 5.0), both of which have been previously described.…”

Phosphatidylinositol 3,4,5-Trisphosphate Activity Probes for the Labeling and Proteomic Characterization of Protein Binding Partners

“…A reported procedure 40 was used to prepare compound 6. Cbz-Lys(Boc)-OMe ( 5 ) (262 mg, 0.664 mmol) was dissolved in 5 mL methanol.…”

“…The characterization of benzophenone-Lys(Boc)-OMe matches with the literature. 40 Benzophenone-Lys(Boc)-OMe (0.279 g, 0.595 mmol) was then dissolved in 4 mL of trifluoroacetic acid / dichloromethane (v/v 1:1). The reaction was allowed to stir at rt for 2 h and was then concentrated under reduced pressure to yield 6 as a yellowish gum (0.272 g, 100%).…”

Exploring Protein Lipidation with Chemical Biology

“…108 Azide-analogs of diacylglycerol (DAG) have been synthesized and can be incorporated into vesicles for biochemical studies with membrane-binding proteins. 109 Alternatively, metabolic labeling with propargylcholine allows bioorthogonal fluorescence imaging of phosphatidylcholine lipids in mammalian cells and in mice (Fig. 11E).…”