Chemistry Reference
In-Depth Information
Our group has been interested in using click chemistry to facilitate the production
of functionalized lipid probes of use for characterizing protein-membrane-binding
interactions (Fig. 4.5). Our initial report in this area focused on the generation of
DAG probes due to the important roles this lipid has in activating targets such as PKC
isoforms, which are critical to cell growth. We specifically targeted functionalization
at the lipid headgroup, such that the added reporter group would be proximal to the
protein-binding domain. Towards this end, we designed and synthesized azido-DAG
16
as a modular scaffold that was derivatized via CuAAC in the final step of synthesis
through reactions with alkynes of type
17
to produce probes
18a-e
, bearing either
a fluorescence or photoaffinity tag [62]. The resulting probes were evaluated for
PKC
binding through incorporation into liposomes for a surface plasmon resonance
(SPR) assay, during which all probes bound with similar affinity to naturally DAG,
demonstrating the validity of these compounds as probes. Following this initial report,
we extended this approach to develop probes corresponding to another important
signaling lipid, phosphatidic acid (PA) [63]. To do so, reporter groups were introduced
onto protected azido-PA derivative
19
in order to produce probes
20a-d,f-g
.Inthis
case, to validate protein-binding properties, we performed SPR assays using an
individual C2 domain of PKC
, since the full-length protein contains multiple lipid-
binding domains. The results demonstrated that while affinities dropped off somewhat
for probes
20a-d,f-g
compared to synthetic PA, these probes nevertheless bound
PKC
-C2 with high affinity, indicating that this strategy for probe development was
generally effective.
In addition to the prior headgroup-derivatized lipid analogs, click chemistry has
also been beneficial for functionalization of lipid acyl chains. Smith and coworkers
initially exploited CuAAC for the synthesis of PC-derived bolaamphiphiles, which
consist of two lipid headgroups connected by long hydrophobic chains that span the
traditional membrane bilayer [64]. In the synthesis, CuAAC was used to efficiently
produce a range of bolaamphiphiles of type
21
; in this case, through the reaction of PC
derivative
22
containing an alkyne at the terminus of the sn-2 acyl chain with multiple
bis-azide linkers (Fig. 4.6). It should also be mentioned that
22
was conveniently
produced in a chemoenzymatic manner from PC by using phospholipase A2 to remove
the sn-2 acyl chain, followed by coupling with an alkynyl-fatty acid. Following
synthesis, it was observed that bolaamphiphiles of type
21
were not capable of
forming stable vesicles. This was attributed to the high polarity of the triazoles, which
likely reside primarily at the aqueous interface rather than the hydrophobic membrane
core. In fact, upon further investigation, these compounds were found to promote dye
leakage from PC/cholesterol liposomes, indicating that they destabilize membranes.
Solaiman and coworkers reported another study in which click chemistry was
performed using synthetic lipids derivatized within the acyl chains to produce novel
lipid analogs [65]. In this report, 17-azido stearic acid and 3-azido decanoic acid
were initially synthesized as clickable fatty acids. In the latter case, an indirect route
involving the opening of a
-lactam with azide was used to circumvent issues with
the susceptibility of 3-substituted carboxylic acid intermediates to eliminations. With
these azido-fatty acids in hand, CuAAC was then implemented to synthesize multi-
ple dimer lipids through binary combinations of triacylglycerols and/or glycolipids,
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