THE DEVELOPMENT AND APPLICATION OF CLICKABLE LIPID ANALOGS FOR ELUCIDATING AND HARNESSING LIPID FUNCTIONS - Click Chemistry in Glycoscience: New Developments and Strategies - page 77

R 1

N

N

N

R 2

R 1 ,R 2 =N 3 or C CH

FIGURE 4.2 Cartoon for the derivatization of intact liposomes using bioorthogonal

chemistry.

into liposomes containing phosphatidylcholine (PC), phosphatidylglycerol (PG), and
cholesterol. Next, CuAAC was explored for functionalizing membrane surfaces
through reaction with synthetic

-thiomannose-azide conjugate 2 . In these studies,
the authors found that high concentrations of the traditional copper sulfate and sodium
ascorbate CuAAC components (2.3 mM and 51.5 mM, respectively) were necessary
to achieve even modest yields for conjugation (25%). As a result, copper chelation
was exploited to as a strategy for improvement, and the use of the water-soluble ligand
bathophenanthroline disulfonate led to around 80% derivatization within 1 hour, and
almost complete reaction after 24 hours. Importantly, the authors used dynamic light
scattering (DLS) and fluorescence-based dye leakage assays to assess the integrity
of membranes during conjugation. In these studies, changes in liposome size ( 50%

increases in diameter) were observed using traditional CuAAC conditions, indicating
membrane disruption. However, this effect was counteracted through ligand addi-
tion and by controlling the order of reactant addition such that no vesicle leakage
or size changes were observed. Finally, the resulting mannose-decorated liposomes
were found to successfully bind target proteins through the agglutination of the lectin
Concanavalin A.
Also in 2006, Kros and coworkers reported the use of a Forster resonance energy
transfer (FRET) system to characterize click-chemistry-based membrane functional-
ization [55]. In this study, phosphatidylethanolamine (PE) analog 3 , bearing an alkyne,
was incorporated into liposomes composed of PC and also lissamine-rhodamine-
tagged PE as a FRET acceptor tag (Fig. 4.3b). Upon reaction with lysine derivative 4
bearing both an azide as well as an NBD (FRET donor) label, using half an equiva-
lent of copper bromide catalyst, FRET activation was observed through enhancement
of the rhodamine (acceptor) emission signal relative to controls lacking copper.
Fluorescence-based analysis showed that the reaction was complete after approxi-
mately 4 hours. DLS experiments were also used to demonstrate that the sizes of the

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