Design and synthesis of hemithioindigo lipids for photo-controlled membrane fusion
Date
2017-11-03
Authors
Montoya Pelaez, Pedro Jose
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Abstract
The goal of this thesis was to design, synthesize and test a chemical switch for
control of membrane fusion. Control of the shape of the molecules that comprise a
membrane should induce a phase change in the membrane. According to current views
of membrane fusion, the phase change should also facilitate formation of fusion
intermediates hence should provoke membrane fusion. The design thus focused on
synthetic lipid targets that have controllable shape changes. Specifically the
incorporation of the hemithioindigo (HT) photochemical switch into the fatty acid chains
of phospholipids was deemed a solution to the design problem.
The synthesis of four phosphatidylcholine (PC) analogues bearing two
hemithioindigo moieties was accomplished. The successful synthesis starts from
bromophenols. The bromide is extended to a nitrile via the Heck reaction with
acrylonitrile. The thiophenol is converted to a thioindoxyl which is coupled with an
aromatic aldehyde to produce the HT core. “Solventless” hydrolysis of the nitrile
produces a carboxylic acid that can be coupled to a phosphoglycerol to give the target
lipids. The synthetic process is both efficient and modular. All new compounds were
characterized by NMR, MS and elemental analysis.
The photochemistry of various HT derivatives was studied to confirm the
expected photoisomerization in both homogenous solutions and vesicle bilayers.
Although the UV-Vis spectra become rather insensitive to the presence of different
isomers, there is evidence to confirm the Z-E switching in a range of organic solvents and in vesicles. Apparent bleaching of the HT-Iipid may indicate a photochemical
dimerization reaction although isomerization would also be consistent with the data.
Fusion was explored by manufacturing PS vesicles with varying concentrations
and isomers of HT-lipid, and was monitored with the Terbium/Dipicolinic acid aqueous
contents mixing assay (Tb/DPA assay). The sensitivity of this assay was lower than
originally expected due to inner filter effects resulting in self-quenching the complex
luminescence. The available data suggest that the synthetic HT-lipids disturb the
membrane structure. Spontaneous fusion, apposition without metal cations, and contents
leakage are some of the observations of the complexity of this system. HT-lipids in one
population of vesicles are able to interact with a second population of vesicles,
presumeably via membrane mixing. These results confirm that shape is a key factor in
the integrity of membranes, and that second generation HT-lipids have the potential to
control membrane fusion.
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Keywords
Membrane fusion, Lipids