THE DEVELOPMENT AND APPLICATION OF CLICKABLE LIPID ANALOGS FOR ELUCIDATING AND HARNESSING LIPID FUNCTIONS - Click Chemistry in Glycoscience: New Developments and Strategies - page 90

imaging and delivery [70]. This was pursued to exploit transport systems that pass
PS across cell membranes in order to deliver the PS analogs and any attached cargo
into cells. In this case, the bioorthogonal tags were incorporated within lipid acyl
chains to avoid the potential of affecting the recognition of the lipid headgroup by
transport machinery. The synthetic strategy entailed incorporation of the tagged acyl
chain onto commercial lysophosphocholine (LPC), followed by Phospholipase D
hydrolysis to PA analogs, and finally introduction of the serine headgroup. While
initial conjugation using CuAAC resulted in low yields (

< 5%), ultrasonication was

found to substantially increase efficacy to 45-90%.
While the design and application of clickable lipids for in vivo applications such
as imaging represents a recent advancement, a number of probe strategies corre-
sponding to different lipids have now been reported. Furthermore, initial labeling
studies have shown promise for the ability to selectively label these compounds in a
native environment, and by doing so, advancing the understanding of the dynamics
of important lipid-based biological processes.

4.5 ACTIVITY-BASED CHARACTERIZATION OF LIPID-BINDING
AND LIPID-MODIFYING PROTEINS

One of the prominent roles of biologically active lipids pertains to their involvement in
noncovalent protein-lipid-binding interactions, which can occur in different settings.
For example, certain lipids act as ligands that trigger the transient docking of soluble
peripheral proteins onto the membrane surface [1,2]. These binding events commonly
regulate both protein function and localization, and have been found to be aberrant
in a number of prominent diseases. In other situations, integral membrane proteins
are known to bind to particular lipids that assist in embedding the protein within
the membrane in particular conformations. In addition, due to the important roles of
lipids, enzymes involved in lipid biosynthesis control important pathways. In recent
years, there has been significant interest in the development of probe-based strategies
that allow for efficient identification and characterization of proteins that bind and
modify lipids. Such studies generally utilize the approach of activity-based protein
profiling, in which probes are employed for the collective labeling, purification, and
analysis of target proteins out of complex samples such as cell extracts, live cells, and
organisms (Fig. 4.9a) [71]. The development of lipid activity probes typically involves
the modification of the ligand structure in order to enforce covalent labeling of target
proteins, and the introduction of a secondary handle that is exploited to label and
manipulate tagged proteins. Since protein-lipid-binding interactions are noncovalent
in nature, photoaffinity tags [72] are commonly implemented to achieve probe-
protein conjugation via photo-cross-linking. In addition, clickable functional groups
have proven invaluable as latent tags for post-derivatization of labeled proteins by
taking advantage of the bioorthogonal nature of this reaction. Using these strategies,
multiple click-chemistry-based lipid activity probes have been reported of late.
In one study of this type, de Kroon and coworkers utilized functionalized PC
probes of type 35a-b to characterize proteins that bind to this lipid (Fig. 4.9b)
[73, 74]. These compounds each included one of a series of photoaffinity moieties