The Synthesis of Amphiphilic Glycolipids as Potential Inhibitors of Bacterial Adhesion

290 Pages·2014·12.23 MB·English

The Synthesis of Amphiphilic Glycolipids as Potential
Inhibitors of Bacterial Adhesion

A thesis submitted to the National University of Ireland in fulfilment of the
requirements for the degree of

Doctor of Philosophy
by
Lorna Abbey, B.Sc.

Department of Chemistry,
National University of Ireland Maynooth,
Maynooth, Co. Kildare, Ireland

September 2013
Research Supervisor: Dr. Trinidad Velasco-Torrijos
Head of Department: Dr. John Stephens
To my parents and Conor,
Thank you for everything.
Contents
Acknowledgements ..................................................................................................................... i
Declaration ................................................................................................................................ iii
Abstract ..................................................................................................................................... iv
Abbreviations ............................................................................................................................. v
Chapter 1: Introduction ....................................................................................................... 1
1.1 Glycolipids and their biological importance ....................................................................... 2
1.2 Glycolipids as potential anti-adhesion agents .................................................................... 2
1.2.1 Anti-adhesion therapy ................................................................................................. 2
1.2.3 The multivalent glycoside effect .................................................................................. 4
1.2.4 Glycoconjugates as inhibitors of Burkholderia cenocepacia adhesion ........................ 6
1.2.5 Glyconjuagates as inhibitors of Pseudomonas aeruginosa adhesion .......................... 8
1.3 Glycolipids as immunomodulators ..................................................................................... 9
1.3.1 The immune response ................................................................................................. 9
1.3.2 Natural Killer T-Cells ................................................................................................... 10
1.3.3 α-Gal-Cer .................................................................................................................... 10
1.3.4 Synthetic analogues of α-GalCer ................................................................................ 11
1.4 Thesis objectives ............................................................................................................... 13
Chapter 2: Alkyl glycolipids as novel soft materials ............................................................. 15
2.1 Amphiphilic glycolipids...................................................................................................... 16
2.2 Gelator molecules ............................................................................................................. 17
2.2.1 Low molecular weight gelators (LMWG) ................................................................... 17
2.2.1.1 The non-covalent interactions involved in gel formation ................................... 18
2.2.2 Non-carbohydrate gelators ........................................................................................ 18
2.2.3 Carbohydrate-based gelators .................................................................................... 19
2.3 Glycomimetics ................................................................................................................... 20
2.3.1 Glycolipid mimetics .................................................................................................... 21
2.3.2 Glycosphingolipid glycomimetics ............................................................................... 22
2.4 Chapter objective .............................................................................................................. 23
2.5 The synthesis of first generation aspartic acid-based β-O-glycolipids 2.26, 2.27 and 2.28
................................................................................................................................................ 25
2.5.1 Initial synthesis of β-O-glycolipid 2.26 ....................................................................... 26
2.5.1.1 Synthesis of galactosyl amine 2.33 ..................................................................... 27
2.5.1.2 Synthesis of acylated aspartic acid glycolipid 2.50 ............................................. 29
2.5.2 Alternative route to form β-O-glycolipid 2.26-2.28 ................................................... 32
2.5.3 Synthesis of β-O glycolipid 2.27 and 2.28 .................................................................. 34
2.6 Synthesis of second generation glycolipids ...................................................................... 36
2.6.1 Synthesis of bivalent β-O-glycolipid 2.29 ................................................................... 36
2.6.2 Synthesis of β-O-glycolipid 2.31 ................................................................................. 37
2.6.3 Synthesis of disaccharide β-O-glycolipid 2.32 ............................................................ 39
2.6.3.1 Synthesis of Lactosyl amine 2.70 ........................................................................ 39
2.6.3.2 Synthesis of aspartic acid building block 2.35 .................................................... 40
2.6.4 Synthesis of C-6 functionalised β-O-glycolipid 2.30 ................................................... 41
2.6.4.1 Attempted synthesis of C-6 functionalised galactose building block 2.83 ......... 43
2.7 Gelation and self-assembly properties of β-O-glycolipids 2.26-2.32 ................................ 47
2.7.1 Gelation and self-assembly properties of first generation of β-O-glycolipids ........... 47
2.7.1.1 SEM on O-glycolipids 2.55, 2.61, and 2.90.......................................................... 51
2.7.1.2 Spectroscopic studies on glycolipid 2.55 and 2.61 ............................................. 55
2.7.2 Gelation and self-assembly properties of second generation β-O-glycolipids .......... 61
2.7.3 Ability of selected O-glycolipids to act as hydrogelators ........................................... 64
2.8 Incorporation of glycolipid 2.55 into a giant unilamellar vesicle (GUV) ........................... 68
2.9 Conclusion ......................................................................................................................... 69
Chapter 3: Synthesis of malonamide glycoconjugates as potential anti-adhesion agents ..... 71
3.1 Introduction ...................................................................................................................... 72
3.1.1 Malonates and their potential application ................................................................ 72
3.1.2 Non-carbohydrate-based malonamides .................................................................... 73
3.1.3 Carbohydrate-based malonamides ............................................................................ 76
3.2 Chapter Objective ............................................................................................................. 77
3.3 The synthesis of malonyl-based glycoconjugates 3.32, 3.33 and 3.34 ............................. 79
3.3.1 Attempted synthesis of β-O-glycoconjugate 3.33 via alkylation followed by amide
coupling ............................................................................................................................... 80
3.3.2 Decarboxylation of malonic acid derivatives ............................................................. 81
3.3.3 Attempted Knoevenagel synthesis of β-O-glycoconjugate 3.43 via alkylation followed
by amide coupling ............................................................................................................... 84
3.3.4 Hydrolysis of alkylidenmalonate 3.48 ........................................................................ 86
3.3.5 Amidation reactions of malonic esters ...................................................................... 87
3.3.6 Malonyl chloride and malonic acid as pecursors for the synthesis of malonamides 88
3.4 Synthesis of O-glycoconjugate 3.33 .................................................................................. 90
3.4.1 DMTMM as an alternative peptide coupling reagent ................................................ 90
3.5 Synthesis of N-glycoconjugate 3.32 .................................................................................. 92
3.5.1 Synthesis of galactosyl amine 3.66 ............................................................................ 92
3.6 Synthesis of Malonyl-based O-glycoconjugate 3.44 ......................................................... 93
3.7 Conclusion ......................................................................................................................... 95
Chapter 4: Synthesis of glycolipids-based on aromatic scaffolds as potential anti-adhesion
agents ............................................................................................................................... 96
4.1 Introduction ...................................................................................................................... 97
4.2 Aromatic-based glycolipids and their biological importance ........................................... 98
4.2.1 Synthesis and application of divalent glycoconjugates ............................................. 98
4.2.2 Synthesis and applications of trivalent glycoconjugates ......................................... 100
4.2.3 Synthesis and applications of oligovalent glycoconjugates ..................................... 102
4.2.4 Synthesis and applications of glycolipids based around an aromatic core ............. 103
4.3 Chapter objective ............................................................................................................ 104
4.3 The synthesis of the first generation of glycolipids 4.38 and 4.39 based on aromatic
scaffolds ................................................................................................................................ 106
4.3.1 Synthesis of O-glycoconjugate 4.38 ......................................................................... 107
4.3.1.1 Initial synthesis of aromatic backbone 4.47 utilising trimesoyl chloride .......... 108
4.3.1.2 Attempted Synthesis of aromatic backbone 4.47 utilising trimesic acid .......... 112
4.3.1.3 Synthesis of glycolipid 4.48 via pentafluorophenyl (PFP) esters ...................... 114
4.3.2 Synthesis of O-glycoconjugate 4.39 ......................................................................... 116
4.3.2.1 Synthesis of aromatic core 4.72 ........................................................................ 116
4.4 The synthesis of first generation triazole containing glycolipids 4.40 and 4.41 based on
aromatic scaffolds ................................................................................................................. 118
4.4.1 Cu catalysed azide-alkyne cycloaddition (CuAAC) ................................................... 119
4.4.2 Synthesis of β-O-glycoconjugate 4.40 ...................................................................... 121
4.4.3 Synthesis of β-O-glycoconjugate 4.41 ...................................................................... 122
4.5 The synthesis of the second generation of glycolipids-based on aromatic scaffolds 4.36
and 4.37 ................................................................................................................................ 123
4.5.1 Synthesis of N-Glycoconjugate 4.36 ........................................................................ 124
4.5.2 Synthesis of N-glycoconjugate 4.37 ......................................................................... 125
4.6 The synthesis of the second generation triazole containing glycolipid 4.42 based on an
aromatic scaffold .................................................................................................................. 126
4.7 The synthesis of second generation glycoconjugate 4.43 based on an aromatic scaffold
.............................................................................................................................................. 128
4.7.1 Initial synthesis of glycoconjugate 4.43 ................................................................... 129
4.7.2 Attempted synthesis of glycoconjugate 4.43 using pentafluorophenyl esters ....... 130
4.7.3 Synthesis of glycoconjugate 4.43 ............................................................................. 131
4.8 The synthesis of monovalent aromatic glycolipid 4.44 .................................................. 132
4.9 The synthesis of non-symmetrical glycolipids based on an aromatic scaffolds 4.116 and
4.117 ..................................................................................................................................... 133
4.9.1 Synthesis of non-symmetrical aromatic glycolipid 4.116 ........................................ 135
4.9.2 Synthesis of non-symmetrical aromatic glycoconjugate 4.117 ............................... 137
4.10 The synthesis of glycoconjugates with more complex carbohydrate epitopes ............ 139
4.10.1 Synthesis of glycosyl donor 4.129 .......................................................................... 140
4.10.2 Synthesis of glycosyl acceptor 4.130 ..................................................................... 141
4.10.3 α-Galactosylation between glycosyl donor 4.129 and glycosyl acceptor 4.130 .... 142
4.11 Spectroscopic analysis on selected glycolipids ............................................................. 145
4.11.1 Concentration studies on first generation glycolipids 4.48 and 4.116 .................. 145
4.11.2 Concentration studies on second generation glycolipids 4.109, 4.125 and 4.126 147
4.12 Conclusions ................................................................................................................... 149
Chapter 5: Evaluation of glycolipids as potential inhibitors of bacterial adhesion .............. 151
5.1 Cystic Fibrosis .................................................................................................................. 152
5.1.1 CFTR protein ............................................................................................................. 152
5.1.2 CF and bacterial infections ....................................................................................... 153
5.2 Burkholderia cencocepacia complex (Bcc) ...................................................................... 153
5.2.1 Virulence of Bcc........................................................................................................ 155
5.3 Bacterial adhesion to cell surfaces .................................................................................. 155
5.3.1 Bcc invasion of epithelial cells .................................................................................. 156
5.4 Chapter Objective ........................................................................................................... 157
5.5 Anti-adhesion assay ........................................................................................................ 158
5.6 Biological evaluation of glycolipids 2.26 and 2.29 based on aspartic acid scaffolds ...... 159
5.7 Biological evaluation of glycolipids 3.32 and 3.34 based on aliphatic scaffolds............. 161
5.8 Biological evaluation of glycolipids based on aromatic scaffolds 4.27-4.34................... 163
5.8.1 Rigid N-glycolipids 4.36 and 4.37 versus flexible O-glycolipids 4.38 and 4.39 ........ 163
5.8.2 1,2,3-Triazole-containing O-glycolipids 4.40 and 4.41 and N-glycolipids 4.42 ........ 166
5.8.3 Biological evaluation of O-glycolipid 4.43 and N-glycolipid 4.44 ............................. 169
5.9. Conclusions based on structural-activity relationship ................................................... 170
5.9.1 Multivalent versus monovalent: .............................................................................. 170
5.9.2 Flexibility versus rigidity: .......................................................................................... 171
5.9.3 Importance of hydrophobic chain ............................................................................ 172
5.10 Conclusions ................................................................................................................... 174
Chapter 6: Experimental details ....................................................................................... 175
6.1 General Procedure and Instrumentation ........................................................................ 176
6.2 Experimental procedures ................................................................................................ 177
6.2.1 Experimental procedures for Chapter 2 .................................................................. 177
6.2.2 Experimental procedures for Chapter 3 .................................................................. 212
6.2.3 Experimental procedures for Chapter 4 .................................................................. 224
6.2.4 Experimental procedures and materials for Chapter 5 ........................................... 261
Bibliography ................................................................................................................... 265
Author Publications………………………………………………………………………………………………………….275
Acknowledgements
Firstly I would like to thank my supervisor Dr. Trinidad Velasco-Torrijos. It has been an
honour to work with you over the last few years and I sincerely thank you for all the
support and encouragement you have given me. To all lecturing staff at NUIM, a huge
thank you for helping me to get to where I am today! To the technical staff, for their
extreme patience when analysising our often difficult samples, hopefully the mass
spectrometer will stay pyridine free for the foreseable future. To Noel for all the
technological advice and for keeping my computer running smoothly. To Dr. John
O’Brien for performing NMR experiements, and to Dr. Siobhan McClean for hosting me
in your lab in ITT.
To all the post graduate students past and present, a huge thank for making the last
few years so enjoyable. I will certainly miss the random conversations and tasty treats
which used to await me in the coffee room. To Rob, my symbiosis sidekick, you have
been there through it all. Thank you for the support, the laughs, and even the abuse.
You really have always been here for me and I couldn’t ask for a truer friend (I am
holding you to that Hebrew prayer!). To Roisin, my evil enantiomer, thank you so much
for everything. You were an immense help right from the start, and certainly kept us all
entertained with your ridiculous sayings and statements. I think I owe you a few nights
B and B for the amount of times you gave me a bed! To Ursula, for keeping me
company on the many late nights, I can truly say I am not just friends with you because
you are Walshy’s missus. To Dec for the endless abuse, hilarious stories and home
cooked meals, Foxy for the cakes, coffee breaks and many chats, John W for the insults
only he could get away it, Lynn for the power ballads, Valeria for highlighting just how
bad my eating habits are and Gillian for coming back to us, don’t let me down with
them carbohydrates!! You really are a great bunch. I also want to thank Ross for
educating me on music and Finno for giving me an interesting internet browsing
history! To the past post graduate students, Colin for his funny impressions, John for
when he was Greg, and Dennis for the quote wall, it has been memborable. To the
Velasco gang Carol, Roisin, Gama and Andrew thank you for everything. Andrew, I only
hope you enjoy your time here as much as I did. To my write up room buddies, Keely,
Wayne, Niall and Haixin, thanks for the advice and chats! To everyone I didn’t mention
personally, thanks a million, I hope you realise how much easier you’s made it.
i
Thanks also to all the post graduate students in ITT, Ruth, Sarah, Minu, Mark, Luke,
Loiuse and Niamh. It is thanks to you that I am now half a biologist, even if it did
initially look doubtful! At least my antics kept you’s entertained.
To my girls Aisling, Michelle, Roisin and Danielle, I am sorry I haven’t been around
much the last year but I promise from now on you will be sick of the sight of me. Thank
you for the distractions and for keeping me sane.
To Conor, what can I say, you have been my rock. Over the last eight years you have
provided me with unending love and encouragement. You have been such an
emotional, physical and even financial support, and words cannot express how grateful
I am. As a wise women once said “every women should have a Conor in their lives”. I
think a few trips to Old Trafford are definitely in order.
Finally to huge thank you to my family. To my Mam and Dad for their continuing love
and support throughout the years. Even as children you both always encouraged us in
everything we did, and for that, I will be eternally grateful. Oh and don’t worry Dad I
am sure will move out eventually! A huge thank you to Wendy, Gav, Keith and Marion
for always looking out for their little sister, but more importantly for my adorable
nieces and nephews. Cian, Lexi and Craig, you always managed to bring a smile to my
face and truly were the most welcome distraction.

ii
Declaration
I declare that the work presented in this thesis was carried out in accordance with the
regulations of NUI Maynooth. The work is original, except where indicated by reference, and
has not been submitted before, in whole or in part, to this or any other university for any other
degree.

Signed: _______________________________ Date: __________________

iii