A molecular approach towards tethered bilayer lipid membranes [Elektronische Ressource] : synthesis and characterization of novel anchor lipids / Petia Atanasova

Atanaso

Informations

• Publié par: johannes_gutenberg-universitat_mainz
• Publié le: 01 janvier 2007
• Nombre de lectures: 9
• Langue: English
• Poids de l'ouvrage: 7 Mo

Extrait

A molecular approach towards
tethered bilayer lipid
membranes: Synthesis and
characterization of novel
anchor lipids





Dissertation zur Erlangung des Grades
`Doktor der Naturwissenschaft´





am Fachbereich Chemie, Pharmazie und Geowissenschaften
der Johannes Gutenberg Universität Mainz








Petia Atanasova
Geboren in Pleven, Bulgarien





Mainz, April 2007




































Tag der mündlichen Prüfung: 06.06.07


Contents


1. 1. Introduction…………………………………………………………………………1
1.1. The cell membranes…………………………………………………………..…1.. ..
1.2.Model lipid membranes…………………………………………………………..2.
1.2.1. Vesicles……………………………………………………………….… 2
1.2.1. Black lipid membranes………………………………………………..3
1.2.3. Membranes on solid supports………………………………….……..4
1.2.3.1. Supported Bilayer Lipid Membranes………………………4.
1.2.3.2. Tethered Bilayer Lipid Membranes…………………….….5
1.3. Motivation……………………………………………………………...............6.
Literature……………………………………………………………………..…8
2. Synthesis of thiolated lipids…………………………………………………… 10
2.1. Characterisation methods…………………………………………………… 10
2.1.1. NMR…………………………………………………………………… 10
2.1.2. FD-MS………………………………………………………………….11
2.1.3. ATR……………………………………………………………………..11
2.1.4. TGA, DSC ……………………………………………………………… 1.1
2.1.5. Materials …………………………………………………………………12.
2.2. Synthesis of lipids with a different length in the tethering part
(DPTT, DPHT and DPOT)……………………………………………………1..3
2.2.1. Introduction………………………………………………………………13
2.2.2. Motivation…………………………………………………………….. 16
2.2.3. Results and discussion……………………………………………….18
2.2.4. Conclusion………………………………………………………….... 2 1 2.2.5. Experimental part………………………………………………………2. 2
2.3. Synthesis of lipid with longer tethering part…………………………………2.9
2.3.1. Introduction and motivation……………………………....................29
2.3.2. Results and discussion……………………………………………….31
2.3.3. Conclusion………………………………………………………….... 3 3
2.3.4. Experimental part………………………………………………….……33.
2.4. Synthesis of lateral spacer molecules…….………………………………. .3. 8
2.4.1. Introduction and motivation……………………………………………38
2.4.2. Results and discussion……………………………………………….40
2.4.3. Conclusion………………………………………………………….... 4 1
2.4.4. Experimental part……………………………………………………… 4.2
2.5. Synthesis of lipid with bulky anchor - “self-diluted” molecule (DPHDL)….45
2.5.1. Introduction and motivation……………………………………………45
2.5.2. Results and discussion……………………………………………….48
2.5.3. Conclusion………………………………………………………….... 5 1
2.5.4. Experimental part……………………………………………………… 5.1
2.6. Synthesis of thiolated lipid with extended hydrophobic part (DDPTT)… 57
2.6.1. Introduction and motivation……………………………………………57
2.6.2. Results and discussion……………………………………………….60
2.6.3. Conclusion………………………………………………………….... 6 2
2.6.4. Experimental part……………………………………………………… 6.3
2.7. Synthesis of fluorescent labeled lipids…….……………………………….6 8
2.7.1. Introduction and motivation……………………………………………68
2.7.2. Results and discussion……………………………………………….69 2.7.3. Conclusion………………………………………………………….... 7 6
2.7.4. Experimental part……………………………………………………… 7.7
Literature…………………………………………………………………….. 85
3. Investigation the properties of thiolated lipid monolayers
(DPTT, DPHT, DPOT and DDPTT)……………………………………………..89
3.1. General principles of Langmuir – Blodgett technique………………………89..
3.2. Introduction and motivation…………………………………………………. 93
3.3. Results and discussion……………………………………………………… 94
3.3.1. Influence of the lipid structure…………………………………………9…4
3.3.2. Influence of the temperature……………………………………………97
3.3.3. Investigation of anchor and free lipids mixed monolayers……………98.
3.3.4. Investigation of miscibility of lipid monolayers…………………………10.2
3.3.5. Hysteresis of the pure anchor lipids……………………………………10..5
3.3.6. Relaxation time investigation of the monolayers………………………10.6
3.4. Conclusion……...……………………………………………………………. 107
Literature………………………………………………………………………..108
4. Electrical properties of diluted tBLMs……………………………………… 110
4.1. Introduction………………………………………………………………….. 110
4.2. Characterisation techniques…………………………………………………111
4.2.1. Electrochemical Impedance Spectroscopy……………………………11..1
4.2.1.1. Theory………………………………………………………. 111
4.2.1.2. Measurements……………………………………………... 113
4.2.2. Atom Force Microscopy…………………………………………………11..4
4.2.3. Contact angle……………………………………………………………1…14
4.3. Materials and methods……………………………………………………… 115 4.3.1. Anchor and free lipids, solutions………………………………………1…15
4.3.2. Substrates……………………………….………………………………1…16
4.3.3. Monolayer formation……………………………………………………1…16
4.3.4. Bilayer formation…………………………………………………………117
4.3.5. Incorporation of valinomycin……………………………………………1.1.9
4.4. Results and discussion……………………………………………………… 120
4.4.1. Application of DDPTT, DPHT and DPHDL……………………………120
4.4.2. Membrane formation based on LB-diluted monolayers……………1… 20
4.4.3. Membrane formation of DPOT-based self-assembled monolayers1… 24
4.4.4. Membrane formation of DDPTT-based self-assembled
monolayers………………………………………………………………1…26
4.5. Conclusion……………………………………………………………………. 128
Literature…………………………………………………………………….. 130
5. Conclusion and outlook…………………………………………………………1.31

Abbreviations……………………………………………………………………...1.35
Curriculum vitae ………………………………………………………………….139





1 1. Introduction
1. Introduction

1.1. The cell membranes

Cells are the fundamental building block of all living organisms. They are as well as
different organelles in the eukaryotic cells surrounded and protected by a membrane.
The study of the cell membrane has a long and extensive history. First, W. Pfeffer in
1877 has discovered that the cell is surrounded by a discrete, but invisible semi-
1permeable membrane. Around 20 years later, E. Overton proposed that the
2membrane consists of an oily or lipid-like substance. In 1910, Höber has shown that
the membrane possesses a high electrical resistivity although the cytoplasm in the
3cell has a high conductivity. A substantial effort has been made last century to gain
more information about structure and function of the cell membrane.

fibers of extracellular matrix carcohydrateplasma cell
membrane
glycoprotein
glycolipid
eeennndddoooppplllaaasssmmmiiiccc
reticulum
hydrophobic
tail
nucleus
hydrophilic
head
golgi
integral protein
mmmiiitttoooccchhhooonnndddrrriiiaaa
ppppppppppeeeeeeeeeerrrrrrrrrriiiiiiiiiipppppppppphhhhhhhhhheeeeeeeeeerrrrrrrrrraaaaaaaaaallllllllll pppppppppprrrrrrrrrrooooooooootttttttttteeeeeeeeeeiiiiiiiiiinnnnnnnnnnrrriiibbbooosssooommmeee lllyyysssooosssooommmeee cholesterollipid bilayer


Figure 1.1. Schematic representation of a biological cell, cell membrane and
phospholipid

The membrane forms a barrier for most molecules and even ions. Water is almost
the only polar molecule able to pass easily. A membrane is mainly composed of
lipids and proteins. The lipids are amphoteric molecules (Figure 1.1) with a polar
hydrophilic “head” attached via an ester or ether bond to two non polar hydrophobic
fatty acid tails. In an aqueous environment, they assemble into a double layer
(bilayer) to form the membrane. The hydrophobic fatty tails face the inside of the
membrane, while the hydrophilic head points outwards. The assembled bilayer
2 1. Introduction
-2behaves like a low dielectric material with capacitance 0.5-1 µF cm . The diverse
functions of the membrane are primarily due to associated proteins. They can be
structural and functional, and depending on the degree of association can be divided
in two groups. Membrane bound proteins are strongly associated or bound and
function on one side of the membrane. The second group includes proteins partly
inserted into the membrane, or traverses the membrane as channels from the
outside to the inside of the cell.
Many models were proposed to explain the organization of the proteins in the lipid
bilayer structure. The most common is the fluid mosaic model proposed by S.J.
4Singer and G.L. Nicolson in 1972 . It is schematically depicted in Figure