August 2011

Recent developments in the generation and application of lipid probes

Recent developments in lipid synthesis capitalize on bioorthogonal reactions and click chemistry to elucidate biological activities 


The toolbox for functionalized lipid probes has expanded considerably over the past several years due to concurrent advances in the synthesis and application of lipid analogs. These innovations have opened up exciting new avenues for using functionalized lipids to probe biological processes that are lipid dependent.

Synthetic methods

The hydrophobic nature of membranes and membrane-interacting proteins presents difficult challenges in understanding the structure and organization of these systems. Here, Michael Best outlines the recent advances in the development of functionalized lipid probes that have provided valuable tools in revealing the complexity of membranes and membrane proteins.

Synthetic methods for generating complex lipids have come to maturation over the past several years, efforts that have facilitated numerous advances in lipid biology, including the identification of novel roles for phosphatidylinositol polyphosphates, glycophosphatidylinositol anchors, lipid A and glycosphingolipids. The discrete derivatization of lipid structures at varying positions within these molecules provides functionalized lipid probes that can aid in characterizing the biological roles of natural lipids. Indeed, many derivatization strategies have been shown to be effective for introducing beneficial functionality while maintaining biological activity even with relatively simple lipids, such as diacylglycerol (1) and phosphatidic acid (2).

Lipid probe strategies

In the application of lipid probes to biological studies, a number of synthetic modifications have proved fruitful (Fig. 1). These have recently been reviewed (3) and are briefly summarized below. Metabolically stabilized analogs (1a) are useful for avoiding undesirable reactions of molecules that might otherwise succumb to endogenous enzymatic modification that could interfere with investigations into biological function. Caged functional groups (1b) can allow lipid analogs to become activated in response to an external stimulus, such as photo-activation. Such activation allows investigators to probe the spatial and temporal regulation of lipid-mediated biological processes.

Probes bearing reporter tags introduced within the structure also have proved popular. Fluorescent dyes (1c) allow for optical imaging of lipids in vivo, permitting the identification of subcellular localization. Photoaffinity tags (1d) can be used to crosslink lipids to proximal proteins, which enables the study of interactions between lipids and proteins that do not otherwise involve covalent bonds. In addition, spin labels (1e) can be introduced to analyze the chemical environment around lipids, conveniently allowing investigators to determine, for instance, the depth of penetration of a lipid component into the membrane bilayer. Finally, biotinylated probes (1f) are effective for affinity purification or surface immobilization.

Bioorthogonal labeling

Recently, dramatic advances in probe-based biological studies have resulted from bioorthogonal labeling and most notably the use of click chemistry, which involves reactions of azides with alkynes to yield triazole products (4, 5). Bioorthogonal reactions employ paired molecules that are designed to react only with one another, avoiding the threat of unwanted reactions that is posed by functional groups that are common within the cellular milieu. The selective derivatization of a target biomolecule within the extremely complex settings of cell extracts, live cells or living organisms, thus, allows for the study of specific biomolecules in contexts that are chemically complex. Furthermore, bioorthogonal reactions generally employ small functional handles, such as the prototypical azide and alkyne of click chemistry, which limit structural perturbations that could interfere with natural biomolecular functionality.

In combination with lipid-probe strategies, bioorthogonal labeling has enabled recent forays into lipid biology. For example, the proteomic analysis of covalent protein lipidation has been achieved through the incorporation of azide- or alkyne-derivatized acyl chains onto proteins (1g) followed by selective purification and identification (6). In addition, live-cell fluorescence-imaging studies using analogs of phosphatidic acid (7) and phosphatidylcholine (8) have been performed through derivatization of alkyne-tagged lipid analogs (1h) via click chemistry. 

Another type of study that has been facilitated by bioorthogonal chemistry is that of activity-based protein profiling, which employs small molecule probes for the collective characterization of proteins based on function (9). These probes generally bear a latent handle that can be derivatized after protein labeling, often through click chemistry, to selectively characterize only those proteins that interact with the probe (1i). Such studies recently have been advanced using phospholipid analogs to identify and characterize lipid-modifying enzymes (10) and protein-lipid binding interactions (11).

References

1. Smith, M. D., Gong, D., Sudhahar, C. G., Reno, J. C., Stahelin, R. V., and Best, M. D. (2008) Synthesis and convenient functionalization of azide-labeled diacylglycerol analogues for modular access to biologically active lipid probes. Bioconjugate Chem. 19, 1855 – 1863.
2. Smith, M. D., Sudhahar, C. G., Gong, D., Stahelin, R. V., and Best, M. D. (2009) Modular synthesis of biologically active phosphatidic acid probes using click chemistry. Mol. Biosyst. 5, 962 – 972.
3. Best, M. D., Zhang, H. L., and Prestwich, G. D. (2010) Inositol polyphosphates, diphosphoinositol polyphosphates and phosphatidylinositol polyphosphate lipids: Structure, synthesis, and development of probes for studying biological activity. Nat. Prod. Rep. 27, 1403 – 1430.
4. Best, M. D. (2009) Click chemistry and bioorthogonal reactions: unprecedented selectivity in the labeling of biological molecules. Biochemistry 48, 6571 – 6584.
5. Sletten, E. M., and Bertozzi, C. R. (2009) Bioorthogonal chemistry: Fishing for selectivity in a sea of functionality. Angew. Chem., Int. Edit. 48, 6974 – 6998.
6. Charron, G., Wilson, J., and Hang, H. C. (2009) Chemical tools for understanding protein lipidation in eukaryotes. Curr. Opin. Chem. Biol. 13, 382 – 391.
7. Neef, A. B., and Schultz, C. (2009) Selective fluorescence labeling of lipids in living cells. Angew. Chem., Int. Ed. 48, 1498 – 1500.
8. Jao, C. Y., Roth, M., Welti, R., and Salic, A. (2009) Metabolic labeling and direct imaging of choline phospholipids in vivo. Proc. Natl. Acad. Sci. U.S.A. 106, 15332 – 15337.
9. Cravatt, B. F., Wright, A. T., and Kozarich, J. W. (2008)Activity-based protein profiling: From enzyme chemistry. Annu. Rev. Biochem. 77, 383 – 414.
10. Tully, S. E., and Cravatt, B. F. (2010) Activity-based probes that target functional subclasses of phospholipases in proteomes. J. Am. Chem. Soc. 132, 3264 – 3265.
11. Gubbens, J., and de Kroon, A. (2010) Proteome-wide detection of phospholipid-protein interactions in mitochondria by photocrosslinking and click chemistry. Mol. Biosyst. 6, 1751 – 1759.

Michael D. Best (mdbest@utk.edu) is an assistant professor of bioorganic chemistry at the University of Tennessee-Knoxville. 


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