investigation of glycolipids self-assemblies using fluorescence spectroscopy and small-angle x-ray

219 Pages·2013·6.37 MB·English

INVESTIGATION OF GLYCOLIPIDS SELF-ASSEMBLIES
USING FLUORESCENCE SPECTROSCOPY AND
SMALL-ANGLE X-RAY SCATTERING
NOOR IDAYU BINTI MAT ZAHID
THESIS SUBMITTED IN FULFILMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
DEPARTMENT OF CHEMISTRY
FACULTY OF SCIENCE
UNIVERSITY OF MALAYA
KUALA LUMPUR
2013
UNIVERSITI MALAYA
ORIGINAL LITERARY WORK DECLARATION
Nameof Candidate:NOOR IDAYU BINTI MAT ZAHID (I.C/PassportNo:860518-14-5096)
Registration/MatricNo:SHC090056
Nameof Degree:DOCTOR OF PHILOSOPHY
Title ofProject Paper/ResearchReport/Dissertation/Thesis (“this Work”):
INVESTIGATION OF GLYCOLIPIDS SELF-ASSEMBLIES USING FLUORESCENCE
SPECTROSCOPY AND SMALL-ANGLE X-RAY SCATTERING
Field of Study:PHYSICAL CHEMISTRY
I do solemnly and sincerely declare that:
(1) I am the sole author/writer of this Work;
(2) This Work is original;
(3) Any use of any work in which copyright exists was done by way of fair dealing and for
permitted purposes and any excerpt or extract from, or reference to or reproduction of
any copyright work has been disclosed expressly and sufficiently and the title of the Work
and its authorship have been acknowledged in this Work;
(4) I do not have any actual knowledge nor do I ought reasonably to know that the making of
this work constitutes an infringement of any copyright work;
(5) I hereby assign all and every rights in the copyright to this Work to the University of
Malaya (“UM”), who henceforth shall be owner of the copyright in this Work and that any
reproduction or use in any form or by any means whatsoever is prohibited without the
written consent of UM having been first had and obtained;
(6) I am fully aware that if in the course of making this Work I have infringed any copyright
whether intentionally or otherwise, I may be subject to legal action or any other action as
may be determined by UM.
Candidate’s Signature Date:25/07/2013
Subscribed and solemnly declared before,
Witness’s Signature Date:25/07/2013
Name:RAUZAH HASHIM
Designation:PROFESSOR
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ABSTRACT
Glycolipids are one of the essential components of the cell membranes to
carry out selected biological functions. Even though the study to resolve the
ambiguity of biological membrane processes is continuously pursuit, it is now
generally recognized that the lipids form lyotropic mesophases in the membranes.
These phases are important biologically and technologically, thus our interest to
characterize them. Herein, we investigate the microenvironment of various lipidic
phases formed by mono- and dialkylated synthetic glycolipids using fluorescence
technique. Both single and branched-chain synthetic glycolipids were observed to
exhibit normal and inverse mesophases respectively. The polarity of different
regions of the hydrophilic head group was estimated on the basis of a parallel study
in different solvents using small fluorescent probes namely tryptophan (Trp) and
two of its ester derivatives (Trp-C and Trp-C ). In contrast, the hydrophobic nature
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of pyrene is expected to favour the tail region of the self-assembly. The ratio of the
two peak intensities (I /I ) in the pyrene fluorescence spectra was used to elucidate
1 3
the local environment of pyrene since it is environmentally sensitive. In addition, the
lifetimes of all fluorescent probes in buffer and lipid were measured. We have also
performed thermotropic and lyotropic investigations on four anomeric-epimeric
related branched-chain glycolipids derived from Guerbet alcohols using small-angle
X-ray scattering to obtain their detailed structural information as well as their partial
binary phase diagram. Similar fluorescence study was carried out for one of these
branched-chain glycolipids, which gave extensive inverse bicontinuous cubic phase.
The results presented here are believed to be important for a better understanding of
the glycolipids fundamental properties which may help uncover some biological
mysteries.
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ABSTRAK
Glikolipid merupakan salah satu komponen utama membran sel untuk
menjalankan fungsi biologikal tertentu. Walaupun kajian bagi merungkai kesamaran
dalam proses membran biologi masih lagi berterusan, kini, umum telah mengiktiraf
bahawa lipid membentuk mesofasa-mesofasa liotropik di dalam membran. Fasa-fasa
ini penting secara biologi dan teknologi, oleh yang demikian, adalah perlu untuk
mencirikannya dalam tesis ini. Kami menyiasat persekitaran mikro pelbagai fasa
lipid yang dibentuk oleh glikolipid sintetik mono- dan dialkil menggunakan teknik
pendafluor. Kedua-dua rantaian tunggal dan bercabang glikolipid sintetik masing-
masing dilihat mempamer mesofasa normal dan songsang. Kekutuban bahagian
berbeza pada kumpulan kepala berkutub telah dianggar berasaskan kajian selari di
dalam larutan yang berlainan dengan menggunakan prob pendafluor kecil iaitu
tryptophan (Trp) dan dua terbitan esternya (Trp-C dan Trp-C ). Sebaliknya, sifat
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hidrofobik pyrene dijangka cenderung kepada bahagian ekor suar penyusunan.
Nisbah keamatan dua puncak (I /I ) pada spektra pendafluor pyrene yang sensitif
1 3
terhadap persekitaran telah digunakan untuk menjelaskan persekitaran setempat
pyrene. Selain itu, masa hayat semua prob pendafluor di dalam bufer dan lipid telah
diukur. Siasatan termotropik dan liotropik juga telah dijalankan bagi empat
glikolipid bercabang terbitan alkohol Guerbet yang berkait secara anomerik dan
epimerik menggunakan penyerakan sinar-X bersudut kecil untuk mendapatkan
maklumat struktur dan gambarajah fasa separa binari sebatian tersebut. Kajian
pendafluor seperti di atas telah dilakukan terhadap salah satu glikolipid rantaian
bercabang yang memberi fasa kubik dwisambungan songsang. Hasil kajian ini
penting untuk pemahaman yang lebih mendalam tentang sifat asas glikolipid yang
mampu membantu mendedahkan beberapa misteri biologikal.
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ACKNOWLEDGMENTS
First of all, I would like to express my utmost gratitude to my supervisor,
Professor Dr. Rauzah Hashim for her guidance, patience and encouragement
throughout my studies and research. Thank you for your motivation and enthusiasm
forscience to follow in the coming days of my research career.
Acknowledgements are also extended to Associate Professor Dr. Osama K.
Abou-Zied from Sultan Qaboos University for his support and continuous
discussions during his supervision on the fluorescence work. I would like to extend
mythanks to Professor Dr. John M. Seddon, Dr. Charlotte E. Conn and Dr. Nicholas
J. Brooks for giving me the opportunities to come and spend time in their labs.
I also would like to thank University Malaya, Sultan Qaboos University, and
CSIRO Manufacturing, Materials & Minerals for providing equipments and facilities for
the research. Not to forget, a special thanks to Dr. Nigel Kirby, Dr. Stephen Mudie
and Dr. Adrian Hawley at the Australian Synchrotron for their assistance.
In addition, I would like to express a big thank you to my colleagues Dr.
Karem Sabah,Associate Professor Dr. Thorsten Heidelberg, Dr. Rusnah Syahila, Dr.
Noraini Ahmad, Seyed Mirzadeh Hosseini, Faramarz Aliasghari Sani, Vijayan
Manickam Achari, Nguan Hock Seng, Sara Ahmadi, Zahrabatoul Mosapour and
Hairul Amani for their help in the equipments and techniques as well as for the
useful discussions.
I am also deeply grateful to my beloved family and friends whose love and
guidance are with me in whatever Ipursue. They are the strongest supporters.
I extend my thanks to Skim Latihan Akademik Bumiputera Fellowship,
Postgraduate Research Grant, Student Exchange Program Grant, OCAR Grant and
High Impact Research Grants (Chancelleryand Faculty) for the financial supports.
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TABLE OF CONTENTS
ABSTRACT iii
ABSTRAK iv
ACKNOWLEDGEMENTS v
TABLE OF CONTENTS vi
LIST OF FIGURES x
LIST OF TABLES xvi
LIST OF SYMBOLS AND ABBREVIATIONS xviii
LIST OF APPENDICES xxi
CHAPTER 1: INTRODUCTION AND LITERATURE REVIEW 1
1.1 Natural Glycolipids 2
1.2 Synthetic Glycolipids 6
1.3 Glycolipids as Liquid Crystal 9
1.4 General Descriptions of Liquid Crystals 10
1.5 Thermotropic Liquid Crystals 14
1.5.1 Monophilic Liquid Crystals 14
1.5.2 Amphiphilic Liquid Crystals 21
1.6 Lyotropic Liquid Crystals 22
1.6.1 Hypothetical Binary Phase Diagram 28
1.6.2 MolecularPacking Parameter 32
1.6.3 Interfacial Curvature 33
1.7 Theoryof Liquid Crystals 36
1.8 Structure-Property Relationship 38
1.8.1 HeadGroup 38
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1.8.2 Linkage 42
1.8.3 Hydrocarbon Chain 43
1.9 Objectives and Overview of Thesis 44
CHAPTER 2: TECHNIQUES AND METHODS 47
2.1 Optical Polarizing Microscopy (OPM) 49
2.1.1 Defect and Texture of Mesophases 52
2.1.2 Thermotropic Phase Behaviour 59
2.1.3 Lyotropic Phase Behaviour 59
2.2 Differential Scanning Calorimetry (DSC) 60
2.3 Small-Angle X-ray Scattering (SAXS) 62
2.4 Fluorescence 66
2.4.1 Steady-State Fluorescence Spectroscopy 70
2.4.2 Time-Resolved Fluorescence Spectroscopy 71
CHAPTER 3: CHARACTERIZATION OFTHE HEAD GROUP AND
THE HYDROPHOBIC REGIONS OF A GLYCOLIPID 74
LYOTROPIC HEXAGONAL PHASE USING
FLUORESCENT PROBES
3.1 Introduction 75
3.2 Research Methodology 78
3.2.1 Materials 78
3.2.2 Sample Preparation 79
3.2.3 Instrumentation 80
3.3 Results and Discussion 80
3.3.1 Probing the Polar Region of the Lipid 80
3.3.2 Probing the Hydrophobic Region of the Lipid 86
3.4 Conclusions 91
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CHAPTER 4: FLUORESCENCE PROBING OF THE
TEMPERATURE-INDUCED PHASE TRANSITION IN 93
A GLYCOLIPID SELF-ASSEMBLY: HEXAGONAL ↔
MICELLAR AND CUBIC↔ LAMELLAR
4.1 Introduction 94
4.2 Research Methodology 96
4.2.1 Materials 96
4.2.2 Sample Preparation 96
4.2.3 Instrumentation 97
4.3 Results and Discussion 98
4.3.1 Probing thePolar Region of the Lipid 98
4.3.2 Probing the Hydrophobic Region of the Lipid 103
4.4 Conclusions 109
CHAPTER 5: INVESTIGATION ON THE EFFECT OF SUGAR
STEREOCHEMISTRY ON BRANCHED-CHAIN 111
GLYCOLIPIDS SELF-ASSEMBLY BY SMALL-
ANGLE X-RAY SCATTERING
5.1 Introduction 112
5.2 Research Methodology 116
5.2.1 Preparation of Compounds 116
5.2.2 Optical Polarizing Microscopy (OPM) 117
5.2.3 Differential Scanning Calorimetry (DSC) 117
5.2.4 Binary Phase Behaviour 118
5.2.5 Small-Angle X-rayScattering (SAXS) 118
5.2.6 Calculation of Structural Parameters 119
5.3 Results and Discussion 121
5.3.1 Thermotropic Phase Behaviour 121
5.3.2 Lyotropic Phase Behaviour 127
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5.3.3 Anomeric-Epimeric Relationships 145
5.4 Conclusions 147
CHAPTER 6: CHARACTERIZATION OFTHE INVERSE PHASE OF
149
A BRANCHED-CHAIN GLYCOLIPID SELF-
ASSEMBLY USING FLUORESCENT PROBES
6.1 Introduction 150
6.2 Research Methodology 152
6.2.1 Materials 152
6.2.2 Sample Preparation 152
6.2.3 Instrumentation 153
6.3 Results and Discussion 153
6.3.1 Probing the Polar Region of the Lipid 153
6.3.2 Probing the Hydrophobic Region of the Lipid 158
6.4 Conclusions 162
CHAPTER 7: CONCLUSIONS 164
APPENDIX A: DIFFERENTIAL SCANNING CALORIMETRY SPECTRA 169
APPENDIX B: SMALL-ANGLE X-RAY SCATTERING DATA 173
REFERENCES 185
LIST OF SCIENTIFIC CONTRIBUTIONS 196
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LIST OF FIGURES

Figure 1.1 : The cell membrane and its components. Adopted from [11]. 3
Figure 1.2 : A few examples of natural GLs. (a) and (b) are 4
glycosphingolipids, (c) and (d) are glycoglycerolipids and (e) is
a glycosyl phosphopolyprenol. Red box: sphingoid base. Blue
box: glycerol backbone.
Figure 1.3 : A few examples of synthetic GLs. 9
Figure 1.4 : Geometry used for defining the order parameter. Redrawn from 12
[56].
Figure 1.5 : Preferred molecule orientation, (cid:1)(cid:2) within a domain (represented 12
by a loosely-defined boundary).
Figure 1.6 : Molecular structure of a rod-like liquid crystal. Redrawn from 13
[57].
Figure 1.7 : Molecular shapes of monophilic liquid crystals. Redrawn from 14
[60].
Figure 1.8 : The molecular organization of calamatic liquid crystals. The 15
vector, (cid:1)(cid:2) represents the director and k is the layer normal.
Redrawn from [60].
Figure 1.9 : Some examples of compounds that exhibit nematic phase 16
together with their liquid crystals phase behaviours (Cr means
crystal, I stands for isotropic and N denotes nematic phase) [63].
Figure 1.10: Cholesteryl benzoate that exhibits cholesteric phase together 17
with its liquid crystals phase behaviours. (Cr means crystal, I
stands for isotropic and N* denotes chiral nematic phase) [66].
Figure 1.11: Some examples of compounds that exhibit smectic phase 18
together with their liquid crystal phase behaviours (Cr means
crystal, I stands for isotropic, N refers to nematic, SmA denotes
smectic A and SmC implies smectic C phase) [53].
Figure 1.12: A typical chemical structure of discotic mesogens: (a) hexa-n- 19
alkanoates of triphenylene and hexa-n-alkoxytriphenylene (b)
hexakis ((4-octylphenyl)ethynyl) benzene [53].
Figure 1.13: Schematic illustration of discotic liquid crystals. Redrawn from 20
[57].
Figure 1.14: Rod-like polymeric liquid crystal. Redrawn from [53]. 21
Figure 1.15: Structural models of GLs. Redrawn from [19]. 22
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