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Synthesis of a Glycolipid Analogue
Towards the Design of a Biomimetic Cell Membrane
by
Serena Singh
A Thesis
presented to
The University of Guelph
In partial fulfilment of requirements
for the degree of
Master of Science
in
Chemistry
Guelph, Ontario, Canada
© Serena Singh, August, 2012
ABSTRACT
SYNTHESIS OF A GLYCOLIPID ANALOGUE
TOWARDS THE DESIGN OF A BIOMIMETIC CELL MEMBRANE
Serena Singh Advisor:
University of Guelph, 2011 Dr. F.-I. Auzanneau
The synthesis of the three 6”-deoxy-6”-thio glycolipid analogues β-D-Gal-(1→6)-β-D-
Gal-(1→4)-β-D-Glu-(1→OCH2)-[1,2,3]-triazole-1-dodecane, β-D-Gal-(1→4)-β-D-Glu-(1→4)-
β-D-Glu-(1→OCH2)-[1,2,3]-triazole-1-dodecane and β-D-Gal-(1→4)-β-D-Glu-(1→4)-β-D-Glu-
(1→OCH2)-[1,2,3]-triazole-1-octadecane is presented here. Glycosylation at position O-4‟ of a
propargyl cellobioside glycosyl acceptor and position O-6‟ of a propargyl lactoside glycosyl
acceptor with a 6-thio-6-deoxy galactosyl donor gave rise to two unique trisaccharides that in
turn underwent copper-catalyzed azide-alkyne cycloadditions with either 1-azidododecane or 1-
azidooctadecane. The potential for each of these analogues to function as tethers of lipid bilayers
to Au(111) was assessed primarily by differential capacitance experiments. Deposition of a
bilayer of DMPC/cholesterol (70:30) by Langmuir-Blodgett (LB) transfer followed by
Langmuir-Schaefer (LS) touch to a self-assembled monolayer of the O-6‟ linked analogue,
diluted with 1-β-D-thioglucose, failed. This led to simplifying the target architecture to diagnose
the quality of the monolayers. A monolayer of the known monosaccharide 1-octadecane-4-(6-
thio-β-D-galacto-pyranosyloxymethyl)-[1,2,3]-triazole1
prepared by LB transfer was found to
support a lipid monolayer deposited by LS touch and this bilayer had the lowest minimum
capacitance observed of 0.9 µF/cm2. An attempt to produce a bilayer by the same method using
the trisaccharide bearing the C-18 alkane chain failed and this was attributed to high water
solubility, which gave rise to poor organization at the air-water interface. A self-assembled
monolayer of this variant went forward to produce a poor quality bilayer with a minimum
capacitance of 7.1 µF/cm2, which was the lowest value obtained for the trisaccharide series of
analogues.
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Acknowledgements
It is my pleasure to express my gratitude towards my supervisor, Dr. France-Isabelle
Auzanneau, for the invaluable gifts of opportunity and experience. She is a role model as a
researcher for her passion, as an educator for her commitment to her students and as a person for
her integrity.
Special acknowledgements to Dr. Jacek Lipkowski and his research group who act in
collaboration with our group to execute the analytical techniques described here. In particular, I
would like to thank postdoctorate Dr. Zhangfei Su for assisting with the electrochemical
experiments and Ph. D. candidate Mr. Michael Grossutti for helping acquire the PM-IRRAS
data. Credit also goes to postdoctorate Dr. Annia Kycia and Ph. D. candidate Mr. Jay Leitch for
contributing their expertise.
I appreciate the members of my advisory committee, Dr. Jacek Lipkowski and Dr. Adrian
Schwan. My experiences with Dr. Lipkowski taught me how collaboration and the sharing of
ideas can bring about tremendous scientific growth. His innovative vision and drive to push the
envelope are qualities that I have admired. Dr. Adrian Schwan has consistently been an excellent
source of support and guidance. His course in physical organic chemistry inspired me to think
outside of the box and in turn heightened my awareness and perspective as a researcher.
It is an honour and a pleasure to have Dr. William Tam as a member of my examination
committee. I am lucky to have had the opportunity to learn from his wisdom as a student in his
organic synthesis class.
Finally, I would like to thank my past and present colleagues Mr. Adam Forman, Mr.
Mickael Guillemineau, Dr. Jenifer Hendel, Mr. Deng Kuir, Mr. Christopher Moore and Ms.
Gillian Priske for their camaraderie and friendship.
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Table of Contents
Acknowledgements
Table of Contents
List of Figures
List of Schemes
List of Tables
Glossary of Abbreviations
Chapter 1: Introduction …………………………………………………………………….
1.1 Properties of the Cell Membrane ……………………………………..………..
1.2 Membrane Bilayer Models ………………………………………………..…...
1.2.1 The Hybrid Bilayer Model ………………………………………..….
1.2.2 Solid Supported Bilayer Lipid Membranes (sBLM) ………………...
1.2.3 Polymer Supported Lipid Bilayers ………………………………..….
1.2.4 Tethered Bilayer Lipid Membranes (tBLM) ………..…………..……
1.2.4.1 Carbohydrate Derived Tethers ……………………….…….
1.3 Applied Techniques for Surface Modification and Analysis …………………..
1.3.1 Modification of Au(111) …………………….……………………….
1.3.1.1 Langmuir-Trough Compression Isotherms ……………..….
1.3.2 Methods for Deposition of Lipid Layers ….………….…….………..
1.3.3 Polarization Modulation Infrared Reflection Absorption
Spectroscopy (PM-IRRAS) ……..................................................................
1.3.4 Cyclic Voltammetry (CV) ………………....……................................
1.3.5 Differential Capacitance (DC) ……………....…….............................
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vii
ix
x
xi
1
2
3
4
5
5
5
7
9
9
11
12
13
13
14
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1.4 Introduction to Carbohydrate Chemistry …...……………………….…………
1.4.1 Mechanism of Glycosylation ….………………........………………..
1.4.1.1 Neighbouring Group Participation ….……………………...
1.4.1.2 The Anomeric Effect ………………...……...……………...
1.5 Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC) ….…………...…...
1.6 Scope of the Thesis ………….……………………….………………………...
Chapter 2: Synthesis of the Glycolipid Analogues …….……………………………..……
2.1 Introduction ………………………………..………………………..………….
2.2 Retrosynthetic Analysis ………………………………………………………..
2.3 Synthetic Strategy ……………………………………………………………...
2.3.1 Synthesis of the Glycosyl Donors Bearing Sulfur at C-6 ...………….
2.3.2 Synthesis of the Glycosyl Acceptor with Access for Glycosylation at
O-6‟ .………………………………………………………………………..
2.3.3 Synthesis of the Glycosyl Acceptor with Access for Glycosylation at
O-4‟ …..…………………………………………………………………….
2.3.4 Synthesis of Protected Trisaccharide 21 ……..………………………
2.3.5 Synthesis of Protected Trisaccharide 27 …..…………………………
2.3.6 Synthesis of the Long Chain Azides …………………………………
2.3.7 Copper-Catalyzed Azide-Alkyne Cycloadditions ……………………
2.3.8 Deprotections to Generate the Glycolipid Analogues ………….……
2.4 Conclusions ……..……………………………………………………………...
Chapter 3: Assembly of the Glycolipid Analogues .……………………………..………...
3.1 Results and Discussion …..…………………………………………………….
15
16
17
18
20
23
25
26
26
27
27
29
31
32
33
34
35
35
37
39
40
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3.1.1 PM-IRRAS Studies …..………………………………………………
3.1.2 Electrochemical Studies …..………………………………………….
3.1.3 Compression Isotherms ..……………………………..………………
3.1.4 Conclusion ..……………………………..…………………………...
Chapter 4: Future Direction .……………………………..………………………………...
4.1 Next Generation Glycolipid Analogue .……………………………..…………
4.2 Exploring New Techniques …………………..………………………………...
4.3 Final Statements …………………..……………………………………………
Chapter 5: Experimental .…………………………………………………………………..
5.1 General Synthetic Procedures ………………………………………………….
5.2 Synthetic Procedures for Chapter 2 ……………………………………………
5.3 Materials and Experimental Methods for Chapter 3 ….…………..………….
References ………………………………………………………………………………….
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48
50
51
52
54
55
56
57
101
104
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List of Figures
Figure 1. In vivo representation of the phospholipid bilayer and an associated glycolipid.
Figure 2. Membrane bilayer models. Vesicles (top left), black lipid bilayers (bottom left),
solid supported bilayers (top right) and tethered bilayers (bottom right).
Figure 3. The hybrid bilayer model.
Figure 4. Application of a tethered bilayer membrane as a biosensor for the analyte (A).
GT represents tethered gramicidin, Gα is mobile gramicidin A and mobile ion channels are
connected to antibody fragments (Fab) by streptavidin (SA) units.
Figure 5. Monosaccharide derived tether molecules.
Figure 6. IR spectra of 35 randomly oriented (ATR) and bound (IRRAS): (a)
hydrocarbon chain stretching vibrations and (b) ring bending modes.
Figure 7. Langmuir-Blodgett transfer of a monolayer onto a hydrophilic surface.
Figure 8. Hyperconjugation between the ny and ζ* orbitals.
Figure 9. Effect of hyperconjugation on molecular orbital energy levels.
Figure 10. Orientation of the dipoles in the axial and equatorial configurations. X is an
electronegative substituent.
Figure 11. Synthetic targets.
Figure 12. Monosaccharides analyzed.
Figure 13. Legend for analogues referred to in this chapter.
Figure 14. IR spectra (hydrocarbon chain region) of analogues (a) 23 and (b) 35
randomly oriented (ATR) and bound (IRRAS).
Figure 15. Example of the tBLM architecture sought after in this thesis.
Figure 16. Simplified bilayer architecture
2
3
4
7
8
8
10
19
19
20
23
24
40
41
42
43
viii
Figure 17. Comparison of the differential capacitance curves of monosaccharide 36
monolayer on Au(111) prepared by LB transfer (top) and self-assembly (bottom) in 0.1
M NaF pH 8.5.
Figure 18. Comparison of the differential capacitance curves of a bilayer on Au(111) in
0.1 M NaF pH 8.5 (inner leaflet: monosaccharide 36 prepared by LB transfer or self-
assembly; outer leaflet: DMPC/cholesterol (70:30)).
Figure 19. Comparison of the differential capacitance curves of trisaccharide 34
monolayer prepared on Au(111) by LB transfer and self-assembly in 0.1 M NaF pH 8.5.
Figure 20. Comparison of the differential capacitance curves of a bilayer on Au(111) in
0.1 M NaF pH 8.5 (inner leaflet: trisaccharide 34 monolayer prepared by LB transfer or
self-assembly; outer leaflet: DMPC/cholesterol (70:30)).
Figure 21. Compression isotherms of the glycolipid analogues 34, 35 and 36 at the
water-air interface.
Figure 22. Next generation glycolipid analogue. Phyt = phytanyl.
Figure 23. STM image of 1-β-D-thioglucose on gold at a potential of +260 mV.
Figure 24. Topography image of a fBLM generated by AFM.
45
45
46
47
48
51
53
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List of Schemes
Scheme 1. Anomerization in solution.
Scheme 2. SN1 mechanism of glycosylation. R is a protecting group, LG is a leaving
group, LA is a Lewis Acid, HOR1 is an acceptor.
Scheme 3. Formation of the β-linked product and the orthoester by assistance from C-2.
Scheme 4. The thermally induced and copper catalyzed 1,3-dipolar cycloadditions.
Scheme 5. Mechanism of the copper-catalyzed azide-alkyne cycloaddition.
Scheme 6. Oxidative coupling to give the 1,4,5-trisubstituted triazole.
Scheme 7. Retrosynthetic analysis of the target glycolipid analogues.
Scheme 8. Synthesis of the bromide glycosyl donor.
Scheme 9. Synthesis of the trichloroacetimidate glycosyl donor.
Scheme 10. Synthesis of the glycosyl acceptor with access for glycosylation at position
O-6.
Scheme 11. Synthesis of the glycosyl acceptor with access for glycosylation at position
O-4‟.
Scheme 12. Glycosylation at position O-6‟ of the propargyl lactoside glycosyl acceptor
20.
Scheme 13. Glycosylation at position O-4‟ of the propargyl cellobioside glycosyl
acceptor 26.
Scheme 14. Synthesis of the hydrocarbon chain.
Scheme 15. Generation of the compounds containing 1,2,3-triazoles by CuAAC.
Scheme 16. Deprotections to give the three unique glycolipid analogues.
16
17
18
20
22
22
27
28
29
30
31
33
34
35
35
36
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List of Tables
Table 1. Variety of conditions tested for glycosylation with glycosyl acceptor 20.
Table 2. Summary of the minimum capacitance values of the glycolipid analogue
monolayers and of the bilayers prepared by LS touch. Inner leaflet = glycolipid
analogues; outer leaflet = DMPC/cholesterol (70:30).
32
43
xi
Glossary of Abbreviations
Ac
AcO
anhyd
aq
ATR-IR
Calcd.
chol
COSY
conc
CSA
CuAAC
CV
DBU
DC
DIPEA
DMF
DMPC
eq/equiv
Gal
Glu
h
HPLC
acetyl
acetate
anhydrous
aqueous
attenuated total reflectance infrared spectroscopy
calculated
cholesterol
correlation spectroscopy
concentrated
camphorsulfonic acid
copper-catalyzed azide-alkyne cycloaddition
cyclic voltammetry
1,8-diazabicycloundec-7-ene
differential capacitance
N,N-diisopropylethylamine
dimethylformamide
1,2-dimyristoyl-sn-glycero-3-phosphocholine
equivalent
galactose
glucose
hour(s)
high performance liquid chromatography
xii
HRESIMS
HSQC
LB
LS
Me
min
NMR
PM-IRRAS
pyr.
rt
SAM
satd
sBLM
tBLM
THF
TLC
TMSOTf
TOCSY
TsOH
tBLM
high-resolution electrospray ionization mass spectra
heteronuclear multiple quantum correlation
Langmuir-Blodgett
Langmuir-Schaefer
methyl
minute(s)
nuclear magnetic resonance
polarization modulation infrared reflection absorption
spectroscopy
pyridine
room temperature
self-assembled monolayer
saturated
supported bilayer lipid membrane
tethered bilayer lipid membrane
tetrahydrofuran
thin layer chromatography
trimethylsilyl trifluoromethansulfonate
total correlation spectroscopy
p-toluenesulfonic acid
tethered bilayer lipid membrane
1
Chapter 1: Introduction
2
1.1 Properties of the Cell Membrane
The cell membrane is critical to the total function of the cell and is defined by a bilayer of
phospholipids.2 These amphiphilic molecules spontaneously arrange to shield their hydrophobic
hydrocarbon chains from the polar aqueous phase and expose their hydrophilic head groups to
the cell‟s interior and exterior. The resultant bilayer, approximately 3 to 4 nm in length3,
functions as a selective barrier between the internal and external environment. It is composed of
lipids that provide the structural support for proteins that facilitate the transport of ions and small
molecules and carbohydrates that are involved in molecular and cellular recognition processes
(Figure 1).
Figure 1. In vivo representation of the phospholipid bilayer and an associated glycolipid.
Reproduced from ref. 2.
Natural membranes exist in electric fields of 107
to 108 V/m and transmembrane
potentials range from -10 to 250 mV.4 Their
insulative character is defined by a resistance of 10
MΩ∙cm2 and a capacitance below 1 µF/cm
2.4
The cell membrane is fluid in nature; therefore, the
rate of lateral diffusion of the constituent lipids is a meaningful parameter. The diffusion
coefficient describing this process has a magnitude of 10-8
cm2/s.
5
3
1.2 Membrane Bilayer Models
The study of the cell membrane is a challenge due to its overwhelming complexity and
thus research has been directed towards designing simplified models of this system that
accurately reflect structure and function in vivo.
The four major classes of bilayer models are vesicles, black lipid bilayers, solid
supported lipid bilayers and tethered lipid bilayers (Figure 2).
Figure 2. Membrane bilayer models. Vesicles (top left), black lipid bilayers (bottom left),
solid supported bilayers (top right) and tethered bilayers (bottom right).
Reproduced from ref. 6.
Vesicles and black lipid bilayers, first employed in the 1960s, are examples of the earliest
constructions of bilayer models. The activity of proteins reconstituted in vesicles has been
studied but the lack of control over the interior concentration of a vesicle limits the quantification
of associated electrical properties.6 Black lipid bilayers are assembled by applying a lipid coat
over a spacer in a Teflon film. With spacious hydrophilic zones on both sides of the bilayer and
electrical properties that mimic natural membranes, these models are particularly useful for the
study of ion channels. The critical disadvantage of this model is that it lacks stability.7 Their
4
sensitivity to mechanical perturbations results in lifetimes of only minutes to hours. Furthermore,
surface techniques cannot be applied to probe structure and function.
The presence of a solid support provides stability and a surface that can be probed by a
wide range of techniques and those applicable to this research will be discussed in detail in the
following sections. Notable techniques that are not discussed here include X-ray and neutron
reflectometry and diffraction8, ellipsometry
8, surface plasmon spectroscopy
9, quartz crystal
microbalance10
and electrical impedence11
spectroscopy. The enhanced stability is important in
the application of these systems on a large scale as biosensors and lab-on-a-chip devices.12
1.2.1 The Hybrid Bilayer Model
The hybrid bilayer model, constructed by Dr. A. L. Plant in 199913
, employs a monolayer
of hydrophobic tails, assembled by binding alkanethiols to a metal support on silicon or glass,
and phospholipids that spontaneously arrange to shield their hydrophobic tails from the polar
aqueous environment (Figure 3). The hybrid bilayer model is stable over months and simple to
prepare but integration of proteins into this rigid structure is a challenge and transport studies are
virtually impossible since the hydrophilic region only exists on one side of the bilayer.
Figure 3. The hybrid bilayer model. Reproduced from ref. 13.
5
1.2.2 Solid Supported Bilayer Lipid Membranes (sBLM)
The solid supported lipid bilayer model is represented by a fluid lipid bilayer that floats
on an aqueous layer. This affords the generation of a hydrophilic region between the support,
constructed from quartz, indium tin oxide or gold, and the bilayer.14
This region is typically only
1 to 2 nm in length such that the reconstitution of proteins with large hydrophilic domains is a
challenge. Interactions of the protein with the support can alter if not inhibit activity and lateral
mobility. Instabilities lead to short lifetimes and structural defects in the bilayer are abundant.
Furthermore, an undefined hydrophilic zone makes electrical measurements difficult to carry out.
1.2.3 Polymer Supported Lipid Bilayers
Polymer supported bilayers represent an improvement of the solid supported lipid
bilayers previously described. In this design, a polymer cushion such as polyethylene glycol
bonded to glass, quartz or silicate mimics the cytoskeleton.15
This approach does compromise the
homogeneity, fluidity and insulative properties of the bilayer, however, the more spacious
hydrophilic region allows for the incorporation of membrane proteins with larger hydrophilic
domains.
1.2.4 Tethered Bilayer Lipid Membranes (tBLM)
The leading design in membrane bilayer models is the tethered lipid bilayer model, in
which the bilayer is anchored to a solid support by a tether that supports hydrophilic zones on
both sides of the membrane. These models are simple to construct and possess long term
stability. The challenge is to design a tether that maintains the fluidity (lipid lateral diffusion of
10-8
cm2/s)
5 and insulative properties (a resistance of 10 MΩ cm
2 and a capacitance below 1 µF
cm-2
)4 of a natural membrane without inhibiting the incorporation of membrane proteins.
6
Tethering systems reported in the literature include polymeric16
and peptide17
tethers.
Closely spaced and short tether give bilayers with the desired high resistance18
however the
incorporation and function of proteins becomes problematic. Tethers that are too long, in
contrast, tend to give bilayers that lack the target electrical properties. Anchoring systems
include thiols, sulfides or disulfides on silver or gold and trichloro- or trimethoxysilanes on oxide
surfaces such as SiO2 or TiO2.16
The density of these tethers and the volume of the aqueous
phase can be varied by diluting the monolayer with polar molecules such as mercaptoacetic acid
disulfide and diethyl disulfide that bind directly to the surface. Tethers carry hydrophobic chains
such as phospholipids, cholesterol derivatives, phytanyl groups and alkyl chains that can be
inserted into the bilayer.7
Valinomycin, a small cyclic ion carrier protein that transports K+ ions, and gramicidin A,
an ion channel for K+ and Na
+, are two model membrane proteins that have been incorporated
into tethered bilayer membranes and studied extensively by electrical impedance spectroscopy to
characterize the conductivity properties of these bilayers.19
Cornel et al.12
developed a biosensor
from a tethered lipid bilayer carrying gramicidin A ion channels conjugated to antibodies
(Figure 4). Application of a potential promotes the flow of ions across the bilayer, however, in
the presence of the analyte, the conjugated antibody crosslinks with its immobilized counterpart
and the ion channel dimer is dislocated. The change in the potential across the membrane is
probed by the gold electrode. Sulfur-gold bonds anchor the bilayer to the surface and membrane
spanning phytanyl lipids found naturally in archaebacteria were selected for insertion into the
bilayer for their excellent insulating and stabilizing effects. The bilayer itself is a mixture of
glycerodiphytanyl ether (GDPE) and diphytanyl ether phosphatidylcholine (DPEPC).
7
Figure 4. Application of a tethered bilayer membrane as a biosensor for the analyte (A).
GT represents tethered gramicidin, Gα is mobile gramicidin A and mobile ion channels are
connected to antibody fragments (Fab) by streptavidin (SA) units. Reproduced from ref. 12.
1.2.4.1 Carbohydrate Derived Tethers
Lipoglycopolymeric tethers have been designed20
but the concept of a purely
carbohydrate based tether system is novel. Such a tethering system would mimic the properties
of the glycocalyx by providing stabilization through a hydrogen bonding network and
maintaining osmotic pressure within the compartment.20
Dr. France-Isabelle Auzanneau designed the first carbohydrate based tether in 2010.1 A
galactose residue was bound to a single-crystalline Au(111) surface by a sulfur atom at position
C-6. Glycolipid analogues 35, 36, 37 and 38 bearing alkyl chains of different lengths (12 and 18
carbon units) at the anomeric position and either glycosidic or 1,4-disubstituted triazole linkages
were synthesized (Figure 5). The corresponding disulfide of 36 was also observed and
characterization by NMR revealed that the signals from the two H-6 protons in the disulfide were
located downfield relative to the same signals in the thiol.
8
Figure 5. Monosaccharide derived tether molecules.
Spectra of the randomly oriented analogues 35 and 37 were acquired by attenuated total
reflectance infrared spectroscopy (ATR-IR) and these were compared to spectra of the bound
analogues acquired by polarization modulation infrared reflection absorption spectroscopy (PM-
IRRAS). These spectra revealed peaks due to asymmetric and symmetric stretching vibrations of
the hydrocarbon chain between 2850 and 3000 cm-1
and these peaks were identified as due to
(from left to right in Figure 6a) vas(CH3), vas(CH2), vs(CH3) and vs(CH2). Ring bending modes
were also observed between 1000 to 1500 cm-1
(Figure 6b).
(a) (b)
Figure 6. IR spectra of 35 randomly oriented (ATR) and bound (IRRAS): (a)
hydrocarbon chain stretching vibrations and (b) ring bending modes. Reproduced from ref. 1.
9
Comparison of the spectra of the randomly oriented analogues (ATR) to the bound
analogues (IRRAS) revealed a decrease in the intensity of vas(CH2) relative to vas(CH3) upon
assembly, which suggested that the molecules oriented perpendicular to the surface.
1.3 Applied Techniques for Surface Modification and Analysis
Passive deposition, Langmuir-Blodgett transfer and Langmuir Schaefer touch are the
three methods of film deposition that were exploited in this work and are described in the
following sections. Also discussed are the techniques that were applied for surface
characterization, which included polarization modulation infrared reflection absorption
spectroscopy (PM-IRRAS), cyclic voltammetry (CV) and differential capacitance (DC). This
approach for deposition and characterization of mono- and bilayers is similar to that adopted by
Dr. Jacek Lipkowski and postdoctorate Dr. Annia H. Kycia in their construction of a floating
bilayer lipid membrane (fBLM) that employed a monolayer of 1-β-D-thioglucose on a gold slide
and the glycolipid GM1 as a tether.21
1.3.1 Modification of Au(111)
Passive self-assembly and Langmuir-Blodgett transfer are the two techniques that were
applied for preparation of the monolayer. Passive deposition of a monolayer21
requires dissolving
the sample in an appropriate solvent and incubating the gold substrate in this solution for 24 h to
facilitate self-assembly. The surface is then washed with solvent to remove any unbound
molecules.
The advantage of the Langmuir-Blodgett (LB) transfer21
for preparation of a monolayer is
that this technique allows for control of the organization and thickness of the film. Deposition of
a monolayer onto a hydrophilic surface first requires orienting the substrate perpendicular to the
water-air interface then submerging it in the aqueous phase. The sample is then dissolved in a
10
water insoluble solvent (for example, chloroform) and this solution is added drop-wise across a
Langmuir trough to produce a floating monolayer known as a Langmuir film. A requirement for
this technique to be applicable is that the molecule must be amphiphilic in nature such that one
end is hydrophilic and the other end is hydrophobic. Following compression of the monolayer to
a target surface pressure, the gold substrate is pulled upwards and through the monolayer such
that the hydrophilic head groups are brought into contact with the surface (Figure 7). The rate of
withdrawal can be controlled to achieve an optimal transfer ratio.
Figure 7. Langmuir-Blodgett transfer of a monolayer onto a hydrophilic surface.
Reproduced from ref. 22.
This method aims to produce homogeneous films but caution must be taken as it does not
guarantee the production of monolayers consisting of all hydrophobic tails oriented
perpendicular to the solid-air interface. He et al.23
demonstrated that 1-triazologalactolipids on
mica surfaces do not orient themselves perpendicular to the air-solid interface and thus produce
thin films if sufficient compression is not invoked prior to Langmuir-Blodgett transfer. Greater
difficulty to transfer a monolayer of 6-triazologalactolipids was encountered because poor
orientation at the solid-air interface resulted in saturation of the surface and thus stacking of the
glycolipids during transfer.
11
1.3.1.1 Langmuir-Trough Compression Isotherms
Compression isotherms are employed to determine the stability and the order of a
monolayer.24
The surface pressure is plotted against the area, which is reduced by mechanical
barriers. The surface pressure is measured by the Wilhelmy plate method. A thin piece of filter
paper is submerged perpendicular to the water-air interface and the force exerted on this plate is
measured. The point at which the monolayer collapses can then be determined by identification
of a sudden decrease in surface pressure.
Phospholipids can adopt highly ordered gel states or fluid liquid crystalline states upon
compression depending on how the hydrocarbon chains are oriented. Assessing the fluidity of
membrane lipids is important in predicting their ability to solubilize membrane proteins. In
general, longer alkyl chains result in stronger intermolecular interactions that are important for
constructing ordered monolayers.25
The stability of the monolayer before deposition can be assessed by observing changes in
the surface pressure over time while the area is held constant. Ideally, the surface pressure should
stay constant but a poor quality monolayer may collapse over time, potentially during the
relaxation time (the time for evaporation of the solvent to occur). The stability of the monolayer
on the trough can be compromised if the water solubility of the constituent molecules is too high.
Examples of glycolipids that are naturally occurring in cell membranes and have demonstrated
low water solubility (10-12
to 10-10
M) include monogalactosyldiglyceride (MGDF) and
digalactosyldiglyceride (DGDG).26
A stable monolayer candidate should thus have an optimal
balance between the size, polarity and hydration capacity of its hydrophilic end and the alkyl
chain length of its hydrophobic end.
12
1.3.2 Methods for Deposition of Lipid Layers
Two different bilayer architectures were explored in this thesis and the methods for the
construction of these are discussed in the following. The lipid samples used in these studies were
mixtures of 70:30 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC):cholesterol (chol)
because this mixture was that which was successfully employed by Dr. Annia Kycia in her thesis
pertaining to the construction of a fBLM.21
Cholesterol typically constitutes 20-40 mol % of
eukaryotic cell membranes and is a vital component because it promotes organization of the
constituent lipids without compromising fluidity.27
It is important to select two lipid components
that are not immiscible to prevent phase separation of the two components on the Langmuir
trough. Two components that are immiscible typically have the same experimental and
calculated average molecular areas at a specified surface pressure but exceptions to this rule do
exist.28
The first model required transfer of only a monolayer to the modified surface and these
experiments were diagnostic of the suitability of the monolayers for incorporation in a tBLM.
Once the monolayer is assembled, another monolayer can be transferred via a Langmuir Schaefer
(LS) touch. The Langmuir Schaefer touch follows the same protocol as the Langmuir-Blodgett
transfer except that in this technique, the substrate is oriented horizontal to the liquid-air
interface. Instead of moving the substrate through the monolayer, the modified surface is gently
brought into contact with the monolayer in the trough. Contact is made with the hydrophobic
ends such that the end result is an architecture analogous to the hybrid bilayer model as
illustrated in Figure 3.
The second model was attempted in aims of constructing a bilayer adjacent to the
modified substrate towards a tBLM type model. Following assembly of the monolayer, the
13
architecture of a tBLM is achieved by carrying out a Langmuir-Blodgett transfer, as described in
the previous section, to assemble the inner leaflet followed by a Langmuir Schaefer touch to
assemble the outer leaflet.29
A bilayer can also be assembled adjacent to a monolayer by vesicle
fusion. Vesicles of defined lipid composition are ruptured on the surface by subjecting them to
osmotic stress and this leads to formation of a bilayer.
1.3.3 Polarization Modulation Infrared Reflection Absorption Spectroscopy (PM-IRRAS)
The orientations of the molecules adsorbed to the surface were elucidated by polarization
modulation infrared reflection absorption spectroscopy (PM-IRRAS). In this experiment, the
polarization of the incident beam is modulated in a perpendicular direction (s-polarization) and a
parallel direction (p-polarization).30
The surface selection rule states that only the p-component
interacts with the surface such that IR bands due to species with perpendicular transition dipole
moments are enhanced and those bands due to species with planar transition dipole moments are
cancelled. The orientation of the molecule with respect to the surface can then be inferred by
comparing the intensities of the observed stretches to an IR spectrum of the randomly oriented
molecules acquired by attenuated total reflectance infrared spectroscopy (ATR-IR).
1.3.4 Cyclic Voltammetry (CV)
Cyclic voltammetry is employed before and after surface modification to detect defects in
the surface and the monolayer and requires the measurement of current at the working electrode
as the electrode potential is cycled.31
Current is generated when a redox reaction occurs at the
surface and this process can either be reversible or irreversible. A positive current peak indicates
an oxidative process and a negative current peak indicates a reductive one.
The peak current, ip, is defined by, ip = (2.69 x 105)n
(3/2)ADo
(1/2)Cov
(1/2), where n is the
number of electrons transferred in the reduction process, A is the area of the electrode, Do is the
14
diffusion coefficient, Co is the concentration and v is the scan rate. If a peak occurs in the CV
curve and the current is proportional to the square root of the scan rate, then the current is due to
a redox reaction occurring at the surface because the process is diffusion controlled. This
relationship can be determined by measuring the current at several different scan rates.
A suitable monolayer should maintain its stability and integrity between -0.40 to +0.45
V.32
Dr. Annia Kycia observed that irreversible reductive desorption of 1-β-D-thioglucose
occurred at -720 mV and phosphate anions from the buffer solution adsorbed to the gold surface
at 292 mV in the presence of the monolayer.21
Integration of the voltammogram to give the total
charge allows for the calculation of the total number of adsorbed molecules based on the number
of electrons transferred per molecule. It is important to note that a discrepancy in the current
maximum can arise when the redox chemistry at the surface is slow relative to the voltage scan
rate. The peak potential can also shift if the process occurring at the surface is irreversible.
1.3.5 Differential Capacitance (DC)
Differential capacitance is another electrochemical technique used to assess the stability
and compactness of mono- and bilayers and to detect defects.21
This analysis requires the
measurement of the capacitance of the electrode as a function of potential.33
The differential capacitance, Cd, is defined as Cd = ((εε0)/d), where ε is the dielectric
constant and d is the thickness of the monolayer. A decrease in capacity thus indicates an
increase in the thickness of the monolayer or an increase in insulative effects. Pin holes in the
film result in an increase in the permittivity of the monolayer and thus an increase in capacitance.
Kycia observed a minimum capacitance of 20 µF∙cm-2
for the unmodified gold surface
that was decreased to 17.3 µF cm-2
upon assembly of the monolayer.21
Capacitative peaks are a
15
result of changes in the surface coverage or changes in the structure of the film. For example,
Kycia observed a peak at -680 mV due to desorption of β-D-thioglucose from the surface.
This experimental technique is particularly useful in determining the effect of varying the
potential on the bilayer. This is important to study since natural membranes exist in electric
fields of 107
to 108 V/m and transmembrane potentials range from -10 to 250 mV.
4
1.4 Introduction to Carbohydrate Chemistry
The interest in carbohydrates as synthetic targets arises from recognition of the
fundamental role they play in biological systems, for example, as energy sources, mediators of
selective immunological processes and structural building blocks. The term „carbohydrate‟
describes their structural formula, Cn(H2O)n, because these compounds are essentially „hydrates
of carbon‟ but this definition has evolved to include variations on this structural backbone.
Carbohydrates include monosaccharides as the fundamental building blocks and can be
expanded to the scale of oligosaccharides and polysaccharides. Aldoses are aldehyde containing
sugars (in constrast to ketoses, which are ketone containing sugars) and the configurations and
conformations of aldohexoses will be the focus of the following.
That which is known about the stereochemical relationship of sugars was first established
by Emil Fischer, who is recognized as the father of carbohydrate chemistry, in the late 1800s.34
D- and L- sugars can be distinguished by determining the configuration of the highest-number
stereocenter (See Scheme 1 for the defined numbering system for carbohydrates). Fischer
projections of D- sugars have the last chiral hydroxyl group oriented to the right.
Hemiacetals are reducing monosaccharides (a term that originates from the ability of
linear D-glucose to reduce Cu2+
to Cu+) that exist in equilibrium between α and β anomeric
16
forms in solution since the possibility of bond reformation from two different modes exists
(Scheme 1).34
The pyranoses (6-membered rings) are generated if OH-5 is the nucleophile and
furanoses (5-membered rings) are produced if OH-4 is the nucleophile. Examples of furanoses
are not shown here because they represent only a minor percentage of the total equilibrium
mixture for the majority of sugars. In the cyclic hexoses, the α-anomer has the hydroxyl group at
C-1 oriented trans to the group attached to the highest number stereocenter (C-5) and the β-
anomer has the opposite configuration. The conformation adopted by D-galactose is a 4C1 chair
conformation that minimizes 1,3-diaxial interactions.
Scheme 1. Anomerization in solution.
1.4.1 Mechanism of Glycosylation
A glycosylation reaction requires a donor (the electrophile), which is equipped with a
good leaving group that can be activated, and an acceptor (the nucleophile) (Scheme 2).34
Glycosyl halides and trichloroacetimidates are examples of the most popular glycosyl donors.
Glycosyl bromides can be activated using mercury salts (Helfrich conditions) or silver salts
(Koenigs-Knorr conditions) while trichloroacetimidate donors are activated using a catalytic
amount of a Lewis acid such as TMSOTf or BF3∙Et2O. A mixture of α and β products are
obtained in a SN1 type mechanism if there is no control over the stereochemical outcome of the
glycosidic linkage. Assistance at C-2 and the anomeric effect are two principles that can be
17
exploited to gear the stereochemistry of the anomeric carbon and these will be discussed in the
following sections.
Scheme 2. SN1 mechanism of glycosylation. R is a protecting group, LG is a leaving group, LA
is a Lewis Acid, HOR1 is an acceptor.
1.4.1.1 Neighbouring Group Participation
Formation of exclusively the 1,2-trans glycosidic linkage in a glycosylation reaction
when an acetate is present at position C-2 is a result of assistance by the C-2 group (Scheme 3).34
The oxacarbenium ion forms following activation of a leaving group by a promotor then
neighbouring group participation leads to the generation of the acetoxonium ion. The formation
of the orthoester is sometimes possible via this intermediate.
18
Scheme 3. Formation of the β-linked product and the orthoester by assistance from C-2.
1.4.1.2 The Anomeric Effect
The preference for the anomeric carbon to position an electronegative substituent axially
rather than in the less sterically hindered equatorial configuration can be explained by the
anomeric effect.
Hyperconjugation of the lone pair on the endocyclic heteroatom (Y) with the ζ*
molecular orbital associated with the bond between the adjacent carbon atom and the exocyclic
heteroatom (C-X) is a stabilizing effect (Figures 8, 9).34
This delocalization of non-bonding
19
electrons requires an anti-periplanar arrangement of the involved orbitals and thus an axial
configuration at the anomeric center.
Figure 8. Hyperconjugation between the ny and σ* orbitals.
Figure 9. Effect of hyperconjugation on molecular orbital energy levels.
The second explanation of the anomeric effect is electrostatic in nature. In the axial
configuration, the dipoles of both heteroatoms are opposing and repulsion is minimized (Figure
10).35
Figure 10. Orientation of the dipoles in the axial and equatorial configurations. X is an
electronegative substituent.
20
1.5 Copper-Catalyzed Azide-Alkyne Cycloaddition (CuAAC)
The thermally induced (non-catalyzed) Huisgen 1,3-dipolar cycloaddition gives a 1:1
mixture of both the 1,4- and 1,5-triazoles because of the energetic similarities between the
respective highest occupied and lowest occupied molecular orbitals (Scheme 4).36
Scheme 4. The thermally induced and copper catalyzed 1,3-dipolar cycloadditions.
Sharpless defined click chemistry in 2001 to encapsulate processes that are reliable,
stereospecific, high yielding and versatile.37
Click chemistry of the copper-catalyzed azide-
alkyne cycloaddition type to give exclusively 1,4-disubstituted-1,2,3-triazoles was realized
simultaneously by the research groups of Sharpless38
and Meldal39
in 2002. Copper catalysis
lowers the activation barrier for the process and increases the rate of the reaction by several
orders of magnitude. The impressive utility of this reaction is a result of the ease with which
alkynes and azides can be introduced and the high efficiency and selectivity with which the
products can be obtained. Furthermore, the triazole structure is especially well suited for
applications in biological systems. These moieties cannot be hydrolytically cleaved, oxidized or
reduced under biological conditions, they possess nitrogen atoms at positions two and three that
can participate in hydrogen bonding as weak hydrogen bond acceptors and their large dipole
moment of 5 Debye favours dipole-dipole interactions.40
Click chemistry of this type has been
widely employed in carbohydrate chemistry and the ability to perform these cycloadditions in the
absence of protecting groups is extremely attractive. Notable applications include surface
21
modification for the preparation of microarrays, conjugation to biomolecules and the formation
of glycoclusters.41
Reaction conditions vary by choice of the Cu(I) source, solvent, base and possibly ligand.
Cu(II) of CuSO4 is reduced in situ by sodium ascorbate and the best results are achieved with
solvent systems of water-alcohol mixtures because of the beneficial effect water has on
accelerating the rate of formation of the acetylide-copper complex.42
An alternative reducing
agent is tris(2-carboxyethyl)phosphine (TCEP), which is most often applied in bioconjugations.
Exclusion of oxygen to prevent the oxidation of Cu(I) to Cu(II) is less important when the
reaction is carried out in the presence of a reducing agent. These conditions described promote
the immediate formation of active Cu(I) clusters while in the case of CuI and CuBr as Cu(I)
sources, an amine base such as N,N-diisopropylethylamine (DIPEA) or 2,6-lutidine (Lut) is
required to promote complexation with the acetylide anion since these compounds form stable
clusters. CuI functions optimally in solvents of medium polarity such as THF, acetonitrile or
DMSO. Ligands such as tris[(1-benzyl-1H-1,2,3-triazol-4-yl)methyl)]amine (TBTA) and
histidine can be employed to protect Cu(I) from oxidation to yield Cu(II) and reactive oxygen
species and from disproportionation to give Cu(0) and Cu(II). They can also increase the reaction
rate by promoting the most active Cu(I) clusters and can function as bases but purification then
becomes a challenge.
The currently accepted mechanism for the formation of the triazole by Cu(I) catalyses
was elucidated by Dr. V. D. Bock in 2006 (Scheme 5).43
Copper, in equilibrium with its dimer
(the ligand in this case is the solvent or the base), forms a π-complex with the alkyne and the pKa
of the terminal hydrogen decreases. Copper insertion decreases electron density at the site of the
alkyne such that, following activation of the azide by the second copper atom, the metallocycle
22
forms as illustrated. Transannular interactions from the lone pair on the nitrogen atom and the
empty molecular orbital of the carbon-copper bond drives the subsequent ring contraction to
form the 1,4-disubstituted-1,2,3-triazole. The final step is regeneration of the catalytic species.
The exact coordination of the acetylide to Cu(I) is still under debate. Particularly complex is
understanding the role of the ligand and fitting this model to the observed second order kinetics
with respect to the alkyne.43
Scheme 5. Mechanism of the copper-catalyzed azide-alkyne cycloaddition.
The 1,4,5-trisubstituted triazole is a potential byproduct that can be produced when Cu(I)
acetylides behave as electrophiles towards the newly formed triazoles still coordinated to Cu(I)
(Scheme 6).42
Oxidative coupling can also occur between Cu(I) acetylides. Dilution and using an
excess of reducing agent can discourage the formation of these generally quantitatively irrelevant
byproducts.42
Scheme 6. Oxidative coupling to give the 1,4,5-trisubstituted triazole.
23
The capability of Cu(I) to poison a reaction by generation of reactive oxygen species has
already been discussed. Excess ascorbate in the presence of copper can also generate these
radicals, which can harm biomolecules, but compounds such as aminoguanidine and
pyridoxamine have been utilized to intercept these radicals.44
Furthermore, the oxidation of
ascorbate yields dehydroascorbate, which is an electrophile that has been reported to react with
protein side chains. This reaction can tolerate most functional groups and rates are not highly
sensitive to steric and electronic effects but the cycloaddition can proceed through the thermal
mechanism to give a mixture of products if strongly activated acyl azides or sulfonyl azides are
involved.45
1.6 Scope of the Thesis
The total synthesis of glycolipid analogues 23, 29 and 34 (Figure 11) is presented in this
thesis.
Figure 11. Synthetic targets.
Chapter 2 discusses the synthetic strategies employed to generate 23, 29 and 34. Chapter
3 outlines the results of the analytical surface and electrochemical experiments that were invoked
to determine the suitability of these analogues as potential tethering molecules. Included in this
24
chapter is a comparison to results obtained for the previously synthesized1 monosaccharides 35
and 36 (Figure 12).
Figure 12. Monosaccharides analyzed.
The direction for future work on this project is set in Chapter 4. Detailed experimental
procedures for the syntheses of 23, 29 and 34 are presented in Chapter 5.
25
Chapter 2: Synthesis of the Glyoclipid Analogues
26
2.1 Introduction
This chapter describes the synthesis of the three glycolipid analogues 23, 29 and 34 that
are designed to tether a phospholipid bilayer to a single-crystalline Au(111) support. Each of
these trisaccharides bears a galactose residue derivatized with a sulfur atom at position O-6”, as
invoked for the monosaccharides presented in M. Guillemineau et al.1 Implementation of a
trisaccharide tether gives rise to an expanded hydrophilic region between the surface and the
bilayer. Furthermore, decoupling the bilayer from the surface is vital for the accommodation of
large transmembrane proteins that extend into the cytoplasm.20
These analogues are each
equipped with a long hydrocarbon chain that is the component intended for insertion into the
bilayer. A 1,2,3-Triazole is the linkage that bridges the hydrophilic and hydrophobic ends.
2.2 Retrosynthetic Analysis
Glycosylation at position O-6‟ of a propargyl lactoside glycosyl acceptor 20 with a
bromide glycosyl donor 7 and at position O-4‟ of a propargyl cellobioside glycosyl acceptor 26
with a trichloroacetimidate glycosyl donor 9 gave rise to two unique trisaccharides. These in turn
underwent copper-catalyzed azide-alkyne cycloadditions with either 1-azidododecane 12 or 1-
azidooctadecane 32 to produce the precursors to the glycolipid analogues 23, 29 and 34 (Scheme
7).
27
Scheme 7. Retrosynthetic analysis of the target glycolipid analogues.
The synthesis of these three novel glycolipid analogues containing 1,4-disubstituted
triazoles that vary in flexibility and chain length is described in the following.
2.3 Synthetic Strategy
2.3.1 Synthesis of the Glycosyl Donors Bearing Sulfur at C-6
The synthesis of the 6-thio-6-deoxy bromide glycosyl donor 7 was carried out in 6 steps
by the procedure reported in M. Guillemineau et al.1 (Scheme 8).
28
Scheme 8. Synthesis of the bromide glycosyl donor.
1,46,47 Reagents and conditions: (a) acetone,
H2SO4, ZnCl2, RT, 21 h; (b) TsCl, acetone, pyr., RT, 6 h; (c) CH3COSK, DMF, 100 °C, 20 h; (d)
H2O: AcOH: TFA (2:3:5), RT, 1.5 h; (e) Ac2O:pyr. (1:1), 50 °C, 1 h; (f) HBr 33% in AcOH,
CH2Cl2, RT, 50 min.
The synthesis of compounds 2 and 3 has been previously reported by Schroeder et al.46
Protection of D-galactose 1 with isopropylidene groups to gain access at O-6 was carried out
using acetone and zinc chloride as the Lewis acid catalyst. Characteristic signals due to the
isopropylidene groups were present in the 1H NMR spectrum of product 2 between 1.25 and 1.48
ppm. Sulfonate ester 3 was then generated from 2 under basic conditions with tosyl chloride. The
displacement of the tosylate by potassium thioacetate was performed in the polar aprotic solvent
DMF at 100 °C under anhydrous conditions to generate compound 4, which carries a sulfur atom
at position O-6 and has been previously described by Cox et al.47
The deprotection of compound 4 to gain access at O-1 was achieved by hydrolysis of the
isopropylidene groups using a mixture of H2O:AcOH:TFA (2:3:5) to obtain hemiacetal 5 as an
α/β mixture (α:β = 1:1). The 1H NMR spectrum of 5 showed a (H-1α, H-2α) coupling constant of
approximately 3.5 Hz and a (H-1β, H-2β) coupling constant of approximately 10 Hz, which is as
expected based on the Karplus relationship.34
Products 5, 6 and 7 were recently described by M. Guillemineau et al.1 An anomeric
mixture of the peracetylated compound 6 was thus obtained after acetylation of hemiacetal 5
29
with acetic anhydride and pyridine at 50 °C. The synthesis of the bromide 7 from peracetylated 6
was performed under a nitrogen atmosphere using hydrobromic acid (33% in acetic acid).
This bromide glycosyl donor 7 was then easily transformed into the trichloroacetimidate
glycosyl donor 9 in two steps (Scheme 9).
Scheme 9. Synthesis of the trichloroacetimidate glycosyl donor. Reagents and conditions: (g)
Ag2CO3, 50% aq acetone, RT, 30 min; (h) Cl3CCN, DBU, CH2Cl2, RT, 3 h.
The hemiacetal 8 was obtained in a 77% yield by stirring the bromide 7 in a mixture of
silver carbonate and aq acetone at rt for 30 min. This hemiacetal 8 was converted to the
trichloroacetimidate glycosyl donor 9 following treatment with trichloroacetonitrile and DBU.
2.3.2 Synthesis of the Glycosyl Acceptor with Access for Glycosylation at O-6’
The synthesis of the propargyl lactoside glycosyl acceptor 20 with a free hydroxyl group
at position O-6‟ is described in Scheme 10.
Treatment of α-lactose with acetic anhydride and pyridine gave peracetylated lactose
14.48
Hasegawa et al.49
reported the synthesis of propargyl lactoside 16 from peracetylated
lactose 14 using BF3∙Et2O as the activating agent and CH2Cl2 as the solvent. Leaving this
reaction for 40 h at rt as described gave the product in only a 20% (impure) yield rather than the
expected 46% literature yield.
30
Scheme 10. Synthesis of the glycosyl acceptor with access for glycosylation at position O-6.
Reagents and conditions: (i) Ac2O, pyr., 50 °C, 1 h; (j) HBr (33% in AcOH), RT, 40 min; (k)
propargyl alcohol, AgOTf, Ag2CO3, CH2Cl2, –10 °C to RT, 18 h; (l) NaOMe/MeOH, RT, 1 h; (m)
PhCH(OMe)2, CSA, MeCN, 70 °C, 2 h; (n) Ac2O, pyr., 50 °C, 1 h; (o) 80% aq AcOH, 100 °C, 45
min.
Propargyl glucosides have been synthesized under Helfrich50
and Koenigs-Knorr51
conditions and so peracetylated lactose 14 was rapidly converted to bromide 15, which has been
previously described by Hronowski et al.48
Treatment of a mixture of bromide 15 and propargyl
alcohol in toluene with Hg(CN)2 at rt produced the desired product, impure, in a 41% yield after
23 h. In contrast, treatment with AgOTf/Ag2CO3 in CH2Cl2 generated propargyl lactoside 16
with a 33% yield when the temperature was increased from –15 °C to RT over 22 h and this
yield was improved to 72% when the reaction was held at –10 °C for 1 h then brought to rt over
the remaining 17 h.
31
The fully protected propargyl lactoside 16 was deprotected under Zemplén conditions to
give disaccharide 17.49
This was then functionalized with a benzylidene acetal following
treatment with benzaldehyde dimethyl acetal and CSA in MeCN at 70 °C for 2 h to give the
novel propargyl lactoside derivative 18.
Following acetylation of 18 to give compound 19, we sought conditions for removal of
the benzylidene acetal to produce diol 20. Disaccharide 19 was first submitted to treatment with
CSA in MeOH at rt over 5 h but this resulted in not only removal of the benzylidene acetal but
also the loss of multiple acetates. In contrast, treatment of 19 with 80% aq AcOH at 100 °C for
45 min successfully produced diol 20 in an 89% yield.
2.3.3 Synthesis of the Glycosyl Acceptor with Access for Glycosylation at O-4’
The synthesis of the propargyl cellobioside glycosyl acceptor with a free hydroxyl group
at C-4‟ is presented in Scheme 11.
Scheme 11. Synthesis of the glycosyl acceptor with access for glycosylation at position O-4’.
Reagents and conditions: (p) AcCl, collidene, CH2Cl2, –35 °C to –5 °C, 2 h; (q) Tf2O, pyr.,
CH2Cl2, –20 °C to RT, 30 min; (r) NaNO3, 50 °C, 20 h.
Selective acetylation of diol 20 with acetyl chloride and the bulky base collidine gave the
desired product 24. Rapid formation of the peracetylated product was observed when the
temperature was raised above –5 °C.
32
The propargyl lactoside 24 then required inversion at C-4‟ to produce the propargyl
cellobioside 26. Various inversion strategies have been reported for carbohydrates: the
Mitsonobu inversion,52
oxidation followed by reduction53
and nucleophilic displacement of a
leaving group.54
Propargyl lactoside 24 was first converted to triflate 25 following treatment with
triflic anhydride in CH2Cl2. No reaction was observed when triflate 25 was heated in water to
reflux but nitrite mediated epimerization55
proved to be successful in producing the propargyl
cellobioside glycosyl acceptor 26. The risk of acetyl migration did not exist because of the cis
relationship between the triflate and the neighbouring ester group.55
This propargyl cellobioside
26 was then submitted to glycosylation at position O-4‟ as illustrated in the next section.
2.3.4 Synthesis of Protected Trisaccharide 21
The glycosylation to produce trisaccharide 21 occurred selectively at position O-6‟ of
diol 20 (Scheme 12). The challenge was in finding conditions that encouraged this glycosylation
to occur in the first place. First the trichloroacetimidate (TCA) 9 and then the bromide 7 were
explored as potential glycosyl donors under a variety of conditions and the results of these
glycosylations with the diol glycosyl acceptor 20 are summarized in Table 1.
Donor Activator Solvent Temperature (°C) Time Yield of 21 (%)
TCA 9 TMSOTf CH2Cl2 –78 to 0 1.5 h 0
BF3∙Et2O CH2Cl2 –10 to 0 1.5 h 0
Bromide 7 Hg(CN)2 MeCN 40 23 h 22, impure
AgOTf CH2Cl2 0 1.5 h 0
AgOTf CH2Cl2 5 to 10 45 min 55
Table 1. Variety of conditions tested for glycosylation with glycosyl acceptor 20.
The trichloroacetimidate glycosyl donor 9 degraded rapidly under activation with
TMSOTf. Performing the reaction at low temperature (–78 °C) in attempts to slow the
degradation kinetics and favor glycosylation was unsuccessful and no product was obtained. The
33
same rapid degradation of glycosyl donor 9 was observed when using BF3∙Et2O as the activating
agent and no glycosylation product could be isolated under these conditions either.
When the bromide glycosyl donor 7 was activated under Helfrich conditions at a reaction
temperature of 40 °C, the desired trisaccharide was obtained, impure, with a low yield of 22%.
This moderate success using the bromide 7 led to exploring activation under Koenigs-Knorr
conditions. It was found that careful control of the temperature under these conditions was the
key to optimizing the yield of trisaccharide 21. When the reaction temperature was held at 0 °C
in the presence of silver triflate, degradation of the bromide 7 was favored over glycosylation
and no product was obtained. However, raising the reaction temperature from 5 to 10 °C under
these same conditions over 45 min was the change required to produce the flexible O-6‟ linked
trisaccharide 21 in a moderate yield of 55% (Scheme 12). It is important to note that the donor
was activated in the presence of excess acceptor in this case, which eased purification of the
product from degraded donor.
Scheme 12. Glycosylation at position O-6’ of the propargyl lactoside glycosyl acceptor 20.
Reagents and conditions: (s) AgOTf, CH2Cl2, 5 °C to 10 °C, 45 min.
2.3.5 Synthesis of Protected Trisaccharide 27
The first attempt to generate the trisaccharide 27 was from the glycosyl acceptor 26 and
the bromide glycosyl donor 7 under activation with 1.5 equiv of Hg(CN)2 at 50 °C in toluene but
no reaction was observed after 18 h. A new strategy involving the trichloroacetimidate glycosyl
donor 9 was then developed. This glycosyl donor 9 was activated with BF3∙Et2O in the presence
34
of the acceptor 26 at rt and the trisaccharide 27 was produced in a poor yield of 22%. Increasing
the temperature to 40 °C and reducing the total reaction time to 1.5 h proved to be the best
conditions for maximizing the yield of 27 while minimizing degradation of the donor (Scheme
13). Leaving the reaction any longer led to increased degradation of the excess donor and thus a
more difficult purification.
Scheme 13. Glycosylation at position O-4’ of the propargyl cellobioside glycosyl acceptor 26.
Reagents and conditions: (t) BF3∙Et2O, CH2Cl2, 40 °C, 1.5 h.
2.3.6 Synthesis of the Long Chain Azides
Following literature procedures,56,57
dodecanol 10 and octadecanol 30 were each treated
with triphenyl phosphine, iodine and imidazole to give iodides 11 and 31, respectively (Scheme
14). Sodium azide was then added to solutions of each of these iodides dissolved in DMF to give
the corresponding known azides 1-azidododecane 12 and 1-azidooctadecane 32.56,57
Scheme 14. Synthesis of the hydrocarbon chain.
Propargyl glycosides 21 and 27 were then paired with azides 12 and 32 as reactive
partners in copper-catalyzed azide-alkyne cycloadditions as described next.
35
2.3.7 Copper-Catalyzed Azide-Alkyne Cycloadditions
The preparation of the glycolipid analogue precursor 22 from trisaccharide 21 and azide
12 by the CuAAC was first attempted using CuSO4∙5H2O and sodium ascorbate (as the reducing
agent) in a mixture of THF/H2O (11:3) at 25 °C but a yield of only 21% was obtained. This yield
of the click product 22 was improved to 59% when the reaction was carried out with CuI as the
Cu(I) source and DIPEA as the base (Scheme 15). These same conditions were successful in
synthesizing the linear trisaccharides 28 and 33 in respective yields of 71% and 70%.
Scheme 15. Generation of the compounds containing 1,2,3-triazoles by CuAAC. Reagents and
conditions: (u) CuI, DIPEA, THF, 25 °C, 20 h; (v) CuI, DIPEA, THF, 25 °C, 18 h.
2.3.8 Deprotections to Generate the Glycolipid Analogues
The final step was the deacetylation of protected trisaccharides 22, 28 and 33 to give
disulfides 23, 29 and 34, respectively (Scheme 16).
36
Scheme 16. Deprotections to give the three unique glycolipid analogues.
We first attempted Zemplén deacetylation to deprotect 22, which was followed by
deionization with DOWEXTM
50WX8-100 resin. A UV active impurity was identified by TLC
that was introduced only after deionization. Following column chromatography to remove the
impurity, only 43% of pure disulfide 23 was obtained. Proton NMR of the isolated impurity
showed signals characteristic of divinylbezene (a constituent of DOWEXTM
ion exchange
resins58
) as well as signals due to disulfide 23. The low pure product yield was thus attributed to
uptake of the product by the resin, which is a phenomenon that has been explained in the
literature.59
Hydrophobic interactions between the long chain of the analogue and the resin were
held responsible for this.
Acid catalyzed transesterification was then explored as an alternative deprotection
strategy. Treatment of 22 with 0.05 M HCl in MeOH failed to remove all of the acetate units.
Gradually increasing the acid concentration to 0.5 M eventually led to degradation of the
trisaccharide. The fully deprotected product could not be obtained under these conditions.
We returned to Zemplén deacetylation as a means to remove the protecting groups but
this time we decided to quench the reaction with a 20% solution of methanolic acetic acid. Two
products were identified by TLC and it was suspected that the major component of the mixture
37
was the disulfide and that the second product was the thiol. To convert the remaining
monosulfide to disulfide, the solvent was evaporated and the residue was dissolved in methanol
and stirred at 40 °C for 20 h. Only one product could be identified by TLC following this time
period as desired. Attempts to separate the sodium acetate salt from the product by means of
Biogel P2 columns eluted with MeOH and 1:1 MeOH:H2O were unsuccessful. For this reason,
we opted for purification by column chromatography to remove the sodium acetate salt. This
approach produced the fully deprotected disulfide 23 in a 75% yield (Scheme 16). The 1H-NMR
spectrum of disulfide 23 shows a multiplet due to the two H-6” protons between 3.00 to 3.03
ppm as was observed for the disulfide of monosaccharide 36 reported in M. Guillemineau et al.1
The characteristic proton of the triazole linkage is also present at 8.00 ppm as expected.
Higher temperature and longer reaction times were required to fully deprotect 28 and 33
to give 29 and 34, respectively (Scheme 16). The same strategy of quenching the reaction
mixture with acetic acid was applied in both cases. Products were stirred in MeOH at 40 °C
overnight to convert any remaining monosulfide to disulfide. The glycolipid 29 was separated
from the sodium acetate salt by column chromatography but difficult purification led to a lower
yield of 70%. The sodium acetate salt was separated from product 34, which carries the more
hydrophobic C-18 chain, by method of extraction from H2O with chloroform. This was followed
by column chromatography and a final yield of 75% was obtained for disulfide 34.
2.4 Conclusions
The bromide glycosyl donor 7 bearing a sulfur atom at position C-6 was synthesized from
D-galactose in 6 steps with an overall yield of 19%. Monosaccharide 7 was converted to the
trichloroacetimidate glycosyl donor 9 in 2 steps. The diol propargyl lactoside glycosyl acceptor
38
20 was synthesized from α-lactose 13 in 7 steps with an overall yield of 23%. Disaccharide 20
was then converted in 3 steps to the propargyl cellobioside glycosyl acceptor 26 that has access
for glycosylation at O-4‟. A wide range of conditions were investigated for producing the O-6‟
linked trisaccharide 21 and it was found that glycosylation of the bromide glycosyl donor 7 with
the propargyl lactoside glycosyl acceptor 20 in the presence of AgOTf as the activating agent
gave rise to the desired product in a 55% yield. The O-4‟ linked trisacharide 27 was generated
from a glycosylation between the trichloroacetimidate glycosyl donor 9 and the propargyl
cellobioside glycosyl acceptor 26 under activation with BF3∙Et2O at 40 °C. These two
trisaccharides 21 and 27 were coupled to 1-azidododecane 12 or 1-azidooctadecane 32 via a
copper-catalyzed azide-alkyne cycloaddition. The protected trisaccharides 22, 28 and 33 were
subsequently submitted to Zemplén conditions to give the respective final glycolipid analogues
23, 29 and 34 as disulfides. The assembly of these analogues to gold surfaces and their potential
as tethering molecules in tBLMs is discussed in Chapter 3.
39
Chapter 3: Assembly of the Glycolipid Analogues
40
3.1 Results and Discussion
The experiments described in the following sections were carried out in collaboration
with Dr. Jacek Lipkowski and his research group. The PM-IRRAS experiments were performed
with assistance by Ph. D. candidate Mr. Michael Grossutti and the electrochemical experiments
required the expertise of postdoctorate Dr. Zhangfei Su. The analogues described in this chapter
are referred to by their compound number (Figure 13).
Figure 13. Legend for analogues referred to in this chapter.
3.1.1 PM-IRRAS Studies
An IR spectrum of 23 self-assembled on gold was acquired by PM-IRRAS and compared
to a spectrum of the randomly oriented analogue acquired by ATR-IR (Figure 14a). A decrease
in the intensity of vas(CH2) relative to vas(CH3) upon assembly, as previously described for
analogue 35 in Section 1.2.4.1 (Figure 14b), was not observed. The conclusion was made that
the orientation of 23 is more disordered than that of 35.
41
(a) (b)
Figure 14. IR spectra (hydrocarbon chain region) of analogues (a) 23 and (b) 35 randomly
oriented (ATR) and bound (IRRAS). Reproduced from ref. 1.
An increase in flexibility is expected especially because the O-6‟ linkage possesses three
degrees of freedom. The monolayer of 29 was also prepared by self-assembly and studied by
PM-IRRAS but no bands could be detected. This was attributed to poor surface coverage or a
patchy analogue film on the surface created by, potentially, mixed orientation of the analogue at
the interface combined with steric effects. These results acted as preliminary evidence that the
best strategy for producing homogeneous monolayers would be by means of LB transfer under
compression.
3.1.2 Electrochemical Studies
The first architecture that was sought after was that of the tBLM (Figure 15). A
monolayer of 23 diluted with commercially available 1-thio-β-D-glucose was produced by self-
assembly on a gold electrode and the quality of this film was evaluated by differential
capacitance measurements. Dilution of the monolayer was deemed a success as the monolayer
minimum capacitance of 12.7 µF/cm2 was lower than that of a SAM of pure thioglucose (14.5
µF/cm2)21
but higher than that of the undiluted SAM (8.6 µF/cm2, Table 2). Unfortunately,
42
deposition of a bilayer by Langmuir-Blodgett (LB) transfer followed by Langmuir Schaefer (LS)
touch failed. There was no observable change in the minimum capacity of the monolayer before
and after modification. The same experimental method to prepare a tBLM failed using the non-
diluted self-assembled monolayer. An attempt to assemble a bilayer by vesicle fusion onto a self-
assembled monolayer of 29 was unsuccessful as well.
Figure 15. Example of the tBLM architecture sought after in this thesis.
The complexity of the architecture was then reduced to deposition of a monolayer of the
analogue by self-assembly or LB transfer followed by deposition of a monolayer of lipids by LS
touch (Figure 16) in order to diagnose the quality of the analogue monolayers. The results of
these experiments are summarized in Table 2 and discussed in the following paragraphs.
43
Figure 16. Simplified bilayer architecture. Inner leaflet = glycolipid analogues; outer
leaflet = DMPC/cholesterol (70:30).
Minimum capacitance (µF/cm2)
Monolayer Bilayer (LS touch)
Analogue Self-assembled LB transferred Self-assembled
monolayer
LB transferred
monolayer
23 8.6 - 8.9 (failed) -
29 8.8 - 8.6 (failed) -
34 15.2 13.0 7.1 12.6 (failed)
35 8.8 9.8 9.2 (failed) 5.6
36 1.1 3.5 1.0 0.9
Table 2. Summary of the minimum capacitance values of the glycolipid analogue monolayers
and of the bilayers prepared by LS touch. Inner leaflet = glycolipid analogues; outer leaflet =
DMPC/cholesterol (70:30).
A monolayer of 23 was prepared by self-assembly and a minimum capacitance of 8.6
µF/cm2 was obtained, which is less than that of bare gold (25 µF/cm
2 at 0.0 V). After deposition
of a monolayer of DMPC/cholesterol (70:30) by LS touch, the minimum capacitance did not
decrease, which suggested that the DMPC/cholesterol monolayer was not present or contained a
vast number of defects. The same experiment was carried out for trisaccharide 29 but a
significant decrease in capacitance following LS touch was not observed in this case either.
These results are not surprising considering that the PM-IRRAS studies revealed that the films
created by 23 are disordered and that the surface coverage of 29 on gold is poor as previously
described. Furthermore, attempts to construct tBLMs from 23 and 29 were unsuccessful.
44
Monosaccharide 35, which is known to orient perpendicular to the surface, was then
explored for comparative purposes. Self-assembly of this analogue gave rise to a monolayer with
a minimum capacitance of 8.8 µF/cm2, which is curiously similar to the values obtained for the
monolayers of trisaccharides 23 and 29. The overall surface coverage of the monosaccharide
monolayer is likely greater than that for the trisaccharide monolayers. The inability to acquire a
PM-IRRAS spectrum of a SAM of analogue 29 is evidence in support of this claim. It is difficult
to make conclusions about the relative thicknesses of the films but the PM-IRRAS spectrum of
the SAM of analogue 23 did indicate significant disorder relative to the SAM of 35. Regardless,
deposition of a lipid monolayer failed to produce a decrease in capacitance.
In response to this finding, a new strategy involving LB transfer for deposition of the
glycolipid monolayer was explored. A surface pressure of 35 mN/m was selected for LB transfer
based on the results of the compression isotherm study, which is presented in a later section. This
method produced a monolayer with a minimum capacitance of 9.8 µF/cm2, which decreased to
5.6 µF/cm2 upon assembly of the bilayer. This decrease in capacitance is evidence for successful
assembly of a bilayer consisting of defects since the desired minimum capacitance of 1 µF/cm2,
observed for natural bilayers, was still not achieved. The poor quality of the bilayer was
attributed to loss of the glycolipid molecules to the aqueous phase during the relaxation period of
the LB transfer procedure.
A monolayer of the more hydrophobic glycolipid monosaccharide 36 was produced via
LB transfer and the resultant bilayer was of excellent quality with a minimum capacitance of 0.9
µF/cm2 (Figures 17, 18). The longer acyl chain also proved to be meaningful in producing a
good quality monolayer by self-assembly and thus a high quality bilayer (minimum capacitance
of 1.0 µF/cm2). The differential capacitance curves of these monolayers and bilayers are flat in
45
the target region (-0.40 V to +0.45 V), which is evidence of a compact monolayer with excellent
surface coverage. Desorption of the monolayer can be observed around -1.1 V.
-1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.40
5
10
15
20
25
30
Self-assembled
LB
C (
F/c
m2)
E (V) vs. SCE Figure 17. Comparison of the differential capacitance curves of monosaccharide 36 monolayer
on Au(111) prepared by LB transfer (top) and self-assembly (bottom) in 0.1 M NaF pH 8.5.
-1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.40
5
10
15
20
C (
F/c
m2)
E (V) vs. SCE
Self-assembled
LB
Figure 18. Comparison of the differential capacitance curves of a bilayer on Au(111) in 0.1 M
NaF pH 8.5 (inner leaflet: monosaccharide 36 prepared by LB transfer or self-assembly; outer
leaflet: DMPC/cholesterol (70:30)).
46
These positive results indicated to us that we may be able to improve the quality of the
trisaccharide monolayers by preparing them via LB transfer. We predicted that stable Langmuir
films would not be produced with the C-12 derivatives due to their high water solubility so the
C-18 trisaccharide derivative 34 was synthesized. The bilayer produced from the self-assembled
monolayer of 34 was found to be higher quality than the bilayer assembled on the LB transferred
monolayer since even this more hydrophobic trisaccharide was too water soluble to uniformly
organize itself at the water-air interface (Figure 19, 20). The DC curve of the monolayer
prepared by LB transfer has peaks in the positive potential region, which indicate pinholes in the
layer that are permitting the adsorption of hydroxide anions on the surface.
-1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.410
15
20
25
30
35
E (V) vs. SCE
C (
F/c
m2)
Self-assembled
LB
Figure 19. Comparison of the differential capacitance curves of trisaccharide 34 monolayer
prepared on Au(111) by LB transfer and self-assembly in 0.1 M NaF pH 8.5.
47
-1.2 -1.0 -0.8 -0.6 -0.4 -0.2 0.0 0.2 0.45
10
15
20
25
30
35
E (V) vs. SCE
C (
F/c
m2)
Self-assembled
LB
Figure 20. Comparison of the differential capacitance curves of a bilayer on Au(111) in 0.1 M
NaF pH 8. (inner leaflet: trisaccharide 34 monolayer prepared by LB transfer or self-assembly;
outer leaflet: DMPC/cholesterol (70:30)).
3.1.3 Compression Isotherms
The compression isotherms of the three analogues 34, 35 and 36 are presented together in
Figure 21. Trisaccharide 34 could be compressed to a maximum surface pressure of 40 mN/m
with no observable collapse in the film pressure. The experimental mean molecular area at any
given pressure was consistently lower than the true value due to the high water solubility of the
trisaccharide. As the surface pressure is increased, a concentration gradient is established at the
air-water interface causing the molecules to diffuse into the water subphase. Monosaccharide 35
could be compressed to a surface pressure of 46 mN/m before it collapsed while compound 36
could be compressed to a maximum surface pressure of 70 mN/m. It has been established that
molecules with longer acyl chains can be compressed to higher surface pressures because the
longer hydrocarbon chain promotes stronger hydrophobic interactions and thus greater order.60
48
10 20 30 40 50 60 70 800
10
20
30
40
50
60
70
Compound 35
Compound 36
Compound 34S
urf
ace
Pre
ssure
(m
N/m
)
Mean Molecular Area (A2)
Figure 21. Compression isotherms of the glycolipid analogues 34, 35 and 36 at the
water-air interface.
From these results, we were able to determine a surface pressure that was sufficiently
high enough to produce uniformly packed films, while not exceeding the critical collapse
pressure. An optimal surface pressure of 35 mN/m was decided upon and thus was attained for
each analogue prior to LB transfer onto the gold surface.
3.1.4 Conclusion
The glycolipid analogue that produced the best quality monolayers by both self-assembly
and LB transfer and thus qualifies as a potential tethering molecules is monosaccharide 36.
However, lack of interest in this monosaccharide halted progress in exploring its potential and a
new and improved trisaccharide analogue as described in chapter 4 has been envisioned for the
future. Bilayers were also produced in the cases of monosaccharide 35 (LB transferred
monolayer) and trisaccharide 34 (self-assembled monolayer) but these were of poorer quality. It
can be concluded that monolayers prepared by LB transfer are more likely to be suitable for
49
incorporation in tBLMs than those prepared by self-assembly but that the efficiency of the LB
transfer can be compromised if the molecules possess high water solubility.
50
Chapter 4: Future Direction
51
4.1 Next Generation Glycolipid Analogue
The experiments described in Chapter 3 demonstrated that the monolayer of the
trisaccharide 34 deposited by LB transfer is better orientated for bilayer assembly than the
monolayer prepared by self-assembly. However, the high water solubility of the trisaccharide
was a major barrier to producing monolayers of high quality via this method. The next
generation glycolipid analogue will thus be a disaccharide equipped with diphytanyl chains to
increase the overall hydrophobicity of the molecule (Figure 22). These phytanyl type chains are
also well known to assist in stabilizing and insulating bilayers.12
This new analogue will have the thiol positioned at C-4‟ instead of C-6‟ so that its
orientation with respect to the gold surface will be analogous to that of 1-β-D-thioglucose, which
is known to orient perpendicular to the substrate.21
Positioning the thiol at the anomeric center
could potentially result in cleavage of the carbon-sulfur bond upon assembly to gold so placing
the thiol at the opposite end of the ring should produce a molecule with perpendicular orientation
without the risk of cleavage.
Figure 22. Next generation glycolipid analogue. Phyt = phytanyl.
A disaccharide with the same structure as that shown in Figure 22 but with a hydroxyl
group in replacement of the thiol will also be synthesized and employed to dilute the tethering
molecule on the Langmuir trough. A glycolipid of similar structure is required to prevent phase
separation of the molecules on the trough. The surface will then be washed with an appropriate
solvent and the unoccupied „holes‟ in the monolayer will be filled by incubation with 1-β-D-
thioglucose. If this analogue is successful in tethering a bilayer then its architecture may be
expanded to the trisaccharide scale to further extend the hydrophilic zone.
52
It would also be interesting to eventually explore the difference between monolayers
containing analogues equipped with triazole linkages and monolayers containing analogues
equipped with glycosidic linkages. The effect of the linkage type on the stability of the bilayer
can then be assessed and this information would be used to improve the structures of future
analogues.
4.2 Exploring New Techniques
In this thesis, PM-IRRAS, cyclic voltammetry and differential capacitance experiments
were employed. PM-IRRAS can also be used for characterizing bilayers in that it can reveal the
orientation of the chains and thus assess the overall quality of the bilayer. Techniques that still
need to be explored for further characterization of mono- and bilayers are chronocoulometry,
electrochemical scanning tunneling microscopy (EC-STM) and atomic force microscopy (AFM).
For example, it would be interesting to employ these methods to compare the structures of
monolayers prepared by self-assembly against those prepared by LB transfer or even
electrochemical deposition.
Chronocoulometry requires the measurement of charge over time following application
of a potential.61
Adsorbed species that will react immediately with the surface can then be
distinguished from solution species that must diffuse. The charge density (µC/cm2) is plotted as a
function of potential and the surface concentration (moles/cm2) and thus area (Å
2) per molecule
can be calculated and plotted as a function of potential.
EC-STM will reveal the spatial arrangement of the molecules on the surface with atomic
resolution with the additional capability of observing potential induced changes.62
A voltage is
applied between the tip of the instrument and the sample and structural information is obtained
53
by relating electron tunnelling decay to the distance between the tip and the sample. In the study
of thioglucose, the monolayer arranged in what was described as wavy stripes with stripe widths
of 2.4 +/- 0.2 nm at a potential of +260 mV (Figure 23) and became disordered at -540 mV.21
Figure 23. STM image of 1-β-D-thioglucose on gold at a potential of +260 mV. Reproduced
from ref. 21.
The overall homogeneity of the bilayer can be visualized by tapping mode atomic force
microscopy. Forces experienced by the tip of the instrument are reflected in the oscillation
amplitude of a cantilever and thus related to the features of the surface.21
The roughness, a
parameter that describes the magnitude of deviation in topographical heights, and the
organization of the bilayer can then be assessed from topography images (Figure 24). Variations
in heights can be differentiated to +/- 0.1 nm.63
The thickness of the bilayer can be evaluated
from force-distance curves that are acquired by bringing the tip into contact with the bilayer.21
Figure 24. Topography image of a fBLM generated by AFM. Reproduced from ref. 21.
54
4.3 Final Statements
A tBLM could not be constructed from the trisaccharides synthesized in this thesis but
the work presented here represents an important stepping stone towards the ultimate goal.
Because of the challenges encountered and the lessons learned, a new and improved glycolipid
analogue has been envisioned for the future. Once this analogue is successfully incorporated into
a tBLM, the final frontier will be the incorporation of membrane proteins and beyond this
horizon there exists a world of potential waiting to be unlocked.
55
Chapter 5: Experimental Section
56
5.1 General Synthetic Procedures
1H NMR (300.13, 400.13 or 600.13 MHz) and
13C NMR (75.5, 100.6 or 150.9 MHz) spectra
were recorded with Bruker Avance spectrometers in CDCl3 (internal standard, for 1H residual
CHCl3 δ 7.24; for 13
C CDCl3 δ 77.0) or CD3OD (internal standard, for 1H residual CD2HOD δ
3.30; for 13
C CD3OD δ 49.0). Chemical shifts (ppm) and coupling constants (J, Hz) were
obtained from a first-order analysis of one-dimensional spectra and assignments of protons and
carbon resonances were based on two dimensional 1H-
1H and
13C-
1H correlation experiments as
well as 1D TOCSY experiments. 1H NMR data are reported using standard abbreviations: singlet
(s), doublet (d), triplet (t), quartet (q), multiplet (m) and broadened (b). TLC was performed on
aluminum plates precoated with Silica Gel 60 (250 µm) containing a fluorescent indicator. The
plates were visualized under UV and/or charred with a 10% solution of H2SO4 in EtOH.
Compounds were purified by flash chromatography with Silica Gel 60 (230-400 mesh) unless
otherwise stated. Solvents were distilled and dried according to standard procedures,64
and
organic solutions were dried over Na2SO4 and concentrated under reduced pressure below 40 °C.
Reversed-phase HPLC purifications were carried out on a Prep Nova Pak® HR C18, 6 µm 60 Å
(25 x 100 mm) column using mixtures of MeCN and water as eluant. Optical rotations were
measured at 23 °C on a Rudolph Research Autopol III polarimeter and reported as follows: [α]D
(c in grams per 100 mL, solvent). High resolution electrospray ionization mass spectra (HRESI
MS) were recorded by the analytical services of the McMaster Regional Center for Mass
Spectrometry, Hamilton, Ontario.
57
5.2 Synthetic Procedures for Chapter 2
5.2.1 The Synthesis of 246
: 1,2:3,4-Di-O-isopropylidene-α-D-galactose
(a) Acetone, ZnCl2, H2SO4
D-Galactose 1 (15.00 g, 83.3 mmol) was dissolved in acetone (188 mL) under N2. Zinc chloride
(18.16 g, 133.2 mmol, 1.6 equiv) was added followed by conc H2SO4 dropwise (0.577 mL, 10.8
mmol, 0.13 equiv) and this mixture was stirred at rt for 21 h. A solution of Na2CO3 (2.99 g, 28.2
mmol) in H2O (52 mL) was added then this mixture was filtered under vacuum and washed with
acetone (100 mL). The solvent was evaporated and, following the addition of H2O (67 mL), the
aq layer was extracted with diethyl ether (4 × 120 mL). The combined organic layers were dried
and concentrated to give monosaccharide 2 (15.26 g, 70%) pure as a yellow oil.
1H NMR (400 MHz, CDCl3, 296 K): δ 5.55 (d, 1H, J = 5.1 Hz, H-1), 4.59 (dd, 1H, J = 2.4, 7.9
Hz, H-3), 4.31 (dd, 1H, J = 2.4, 5.0 Hz, H-2), 4.25 (dd, 1H, 1.5, J = 1.5, 7.8 Hz, H-4), 3.85–3.82
(m, 2H, H-5, H-6), 3.75–3.70 (m, 1H, H-6), 1.51, 1.44, 1.31 (4s, 12H, CH3).
13
C NMR (100 MHz, CDCl3, 296 K): δ 109.5 (C(CH3)2), 108.7 (C(CH3)2), 96.3 (C-1), 71.6 (C-
4), 70.8 (C-3), 70.6 (C-2), 68.2 (C-5), 62.2 (C-6), 26.0, 25.9, 25.0, 24.3 (CH3 × 4).
This product has previously been described in the literature.46
58
5.2.2 The Synthesis of 346
: 1,2:3,4-Di-O-isopropylidene-6-O-p-toluenesulfonate-α-D-galactose
(a) TsCl, acetone, pyr.
Tosyl chloride (13.41 g, 70.3 mmol) was added to monosaccharide 2 then this mixture was
dissolved in HPLC grade acetone (32 mL) and anhyd pyridine (20.8 mL) under N2 and stirred at
rt for 6 h. Ice water (180 mL) was introduced followed by CH2Cl2 (180 mL). The organic layer
was washed with ice water (180 mL × 2), dried and concentrated. Chromatography of the crude
product (6:4 EtOAc-hexanes) gave tosylate 3 (16.79 g, 72%) pure as a yellow oil.
1H NMR (400 MHz, CDCl3, 297 K): δ 7.79–7.77 (m, 2H, OTs), 7.32–7.24 (m, 2H, OTs), 5.43
(d, 1H, J = 8.0 Hz, H-1), 4.56 (dd, J = 2.5, 7.9 Hz, 1H, H-3), 4.28–4.26 (m, 1H, H-2), 4.19–4.15
(m, 2H, H-4, H-5), 4.08–4.04 (m, 2H, H-6), 2.42 (s, 3H, OTs), 1.48, 1.32, 1.29, 1.25 (4s, 12H,
CH3).
13
C NMR (100 MHz, CDCl3, 297 K): δ 144.8 (OTs Ar quat), 132.8 (OTs Ar quat), 129.7 (OTs
Ar), 128.1 (OTs Ar), 109.5 (C(CH3)2), 108.9 (C(CH3)2), 96.1 (C-1),70.5 (C-3), 70.4 (C-2), 70. 3
(C-4), 68.1 (C-6), 65.8 (C-5), 26.0, 25.8, 24.9, 24.3 (CH3 × 4), 21.6 (OTs CH3).
This product has previously been described in the literature.46
59
5.2.3 The Synthesis of 447
: 1:2,3:4-Di-O-isopropylidene-6-S-acetyl-6-thio-α-D-galactopyranose
a) CH3COSK, DMF
Potassium thioacetate (7.86 g, 68.8 mmol) was added to tosylate 3 (16.79 g, 40.5 mmol) then this
mixture was dissolved in anhyd DMF (300 mL) under N2 and stirred at 100 °C for 20 h. The
solvent was then evaporated and the residue was dissolved in CH2Cl2 (500 mL) and washed
successively with brine (2 × 500 mL) and water (2 × 500 mL). The combined organic layers
were dried and concentrated. Chromatography of the crude product (18:85 EtOAc:hexanes) gave
product 4 (12.01 g, 93%) pure as a yellow oil.
1H NMR (400 MHz, CDCl3, 296 K): δ 5.48 (d, 1H, J = 5.0 Hz, H-1), 4.58 (dd, 1H, J = 2.4, 7.9
Hz, H-3), 4.27–4.22 (m, 2H, H-2, H-4), 3.84–3.80 (m, 1H, H-5), 3.16–2.97 (m, 2H, H-6), 2.30 (s,
3H, SCOCH3), 1.44, 1.42, 1.32, 1.28 (4s, 12H, CH3).
13
C NMR (100 MHz, CDCl3, 296 K): δ 195.8 (C=O), 109.5 (C(CH3)2), 108.8 (C(CH3)2), 96.5
(C-1), 72.1 (C-4), 70.9 (C-3), 70.5 (C-2), 66.8 (C-5), 30.5 (SCOCH3), 29.7 (C-6), 26.0
(C(CH3)2), 26.0 (C(CH3)2), 25.0 (C(CH3)2), 24.4 (C(CH3)2).
The product has previously been described in the literature.47
60
5.2.4 The Synthesis of 51: 6-S-Acetyl-6-thio-α,β-D-galactopyranose
a) H2O:AcOH:TFA (2:3:5)
Monosaccharide 4 (12.01 g, 37.7 mmol) was dissolved in a mixture of H2O:AcOH:TFA (2:3:5)
(400 mL) and the mixture was stirred at rt for 1.5 h. The reaction was coconcentrated with
toluene (2 × 250 mL) and chromatography of the residue (90:6:4 EtOAc-MeOH-H2O) gave the
hydrolysis product 5 (6.73 g, 75%, α/β ratio 1:1) pure as a white foam.
1H NMR (400 MHz, CD3OD, 297 K): δ 5.09 (d, 1H, J = 3.2 Hz, H-1α), 4.38 (dd, 1H, J = 3.7, 3.8
Hz, H-1β), 3.98 (t, 1H, J = 6.7 Hz, H-5α), 3.83 (d, 1H, J = 1.7 Hz, H-4α), 3.78 (s, 1H, H-4β),
3.75–3.67 (m, 2H, H-2α, H-3α), 3.49–3.48 (m, 1H, H-5β), 3.45–3.39 (m, 2H, H-2β, H-3β), 3.19–
3.02 (m, 4H, H-6α, β), 2.31 (s, 6H, SCOCH3-α, β).
13
C NMR (100 MHz, CD3OD, 297 K): δ 197.2 (C=O), 98.7 (C-1β), 94.2 (C-1α), 75.2 (C-5β),
74.9, 73.5 (C-2β, C-3β), 71.9 (C-4α), 71.2 (C-4β), 71.2, 70.2 (C-2α, C-3α), 70.4 (C-5α), 30.7,
30.6 (C-6α, β), 30.4 (SCOCH3-α, β).
The product has previously been described in the literature.1
61
5.2.5 The Synthesis of 61: 1,2,3,4-Tetra-O-acetyl-6-S-acetyl-6-thio-α,β-D-galactopyranose
a) Ac2O, pyr.
Pyridine (40 mL) followed by acetic anhydride (40 mL) were added to monosaccharide 5 (6.73
g, 28.2 mmol) and the mixture was stirred at 50 °C for 1 h. The reaction was coconcentrated with
toluene (3 × 120 mL) and the residue was dissolved in CH2Cl2 (500 mL). Extraction was carried
out successively with satd aq NaHCO3 (500 mL) and 2 M aq HCl (500 mL) then the aq layers
were re-extracted with CH2Cl2 (500 mL). The combined organic layers were dried and
concentrated to give product 6 (10.90 g, 95%, α/β ratio 1:1) pure as a white foam.
1H NMR (400 MHz, CDCl3, 295): δ 6.33 (d, 1H, J = 4.0 Hz, H-1α), 5.64 (d, 1H, J = 8.3 Hz, H-
1β), 5.50 (d, 1H, J = 2.6 Hz, H-4α), 5.42 (d, 1H, J = 2.8 Hz, H-4β), 5.30–5.27 (m, 3H, H-2α, H-
2β, H-3α), 5.03 (dd, 1H, J = 3.4, 10.4 Hz, H-3β), 4.11 (t, 1H, J = 7.1 Hz, H-5α), 3.83 (t, 1H, J =
7.0 Hz, H-5β), 3.17–2.93 (m, 4H, H-6α, H-6β), 2.31 (s, 3H, SCOCH3), 2.17, 2.16, 2.16, 2.11,
2.02, 1.97 (8s, 24H, OCOCH3).
13
C NMR (100 MHz, CD3OD, 296): δ 194.3, 170.1, 170.0, 169.9, 169.7, 169.3, 168.9 (α,β-C=O),
91.9 (C-1β), 89.6 (C-1α), 73.1 (C-5β), 70.9 (C-3β), 70.1 (C-5α), 68.0 (C-2α or C-3α), 67.7 (C-
4β), 67.5 (C-4α), 67.5 (C-2β), 66.3 (C-2α or C-3α), 30.8, 30.3 (α,β-SCOCH3), 28.1 (C-6α, C-6β),
21.6, 20.8, 20.7, 20.6, 20.6, 20.5 (α,β-OCOCH3 × 8).
This product has previously been described in the literature.1
62
5.2.6 The Synthesis of 71: 2,3,4-Tri-O-acetyl-6-S-acetyl-6-thio-α-D-galactopyranosyl bromide
a) HBr (33% in AcOH), CH2Cl2
Peracetylated monosaccharide 6 (4.21 g, 10.4 mmol) was dissolved in anhyd CH2Cl2 (54 mL)
and HBr (33% in AcOH) (21 mL) under N2 and this mixture was stirred at rt for 50 min. The
reaction was then diluted with CH2Cl2 (450 mL) then washed with satd aq NaHCO3 (2 × 500
mL) and the aq layers were re-extracted with CH2Cl2 (2 × 250 mL). The combined organic layers
were dried and concentrated. Chromatography of the crude product (4:6 EtOAc-hexanes) gave
bromide 7 (2.58 g, 58%) pure as a brown syrup.
1H NMR (400 MHz, CDCl3, 297 K): δ 6.64 (d, 1H, J = 3.7 Hz, H-1), 5.48 (dd, 1H, J = 1.1, 3.2
Hz, H-4), 5.34 (dd, 1H, J = 3.2, 10.6 Hz, H-3), 4.97 (dd, 1H, J = 3.9, 10.6 Hz, H-2), 4.23 (t, 1H,
J = 7.1 Hz, H-5), 3.15 (dd, 1H, J = 7.2, 13.9 Hz, H-6a), 2.95 (dd, 1H, J = 7.0, 13.9 Hz, H-6b),
2.31 (s, 3H, SCOCH3), 2.14, 2.07, 1.97 (3s, 9H, OCOCH3).
13
C NMR (100 MHz, CDCl3, 297 K): δ 194.0, 170.0, 170.0, 169.7 (C=O), 88.2 (C-1), 72.4 (C-5),
68.2 (C-3), 67.7 (C-4), 67.6 (C-2), 30.4 (SCOCH3), 27.8 (C-6), 20.7, 20.6, 20.5 (OCOCH3 × 3).
This product has previously been described in the literature.1
63
5.2.7 The Synthesis of 8: 2,3,4-Tri-O-acetyl-6-S-acetyl-6-thio-α,β-D-galactose
a) Ag2CO3, 50% aq acetone
Bromide 7 (1.72 g, 4.03 mmol) was dissolved in a mixture of acetone (8.8 mL) and water (8.8
mL) then silver carbonate (0.830 g, 3.01 mmol, 0.75 equiv) was added and the reaction was
stirred at rt for 30 min. This mixture was filtered over a pad of Celite® and washed with acetone
(20 mL). The filtrate was combined with CH2Cl2 (100 mL) and washed successively with H2O
(100 mL), satd aq NaHCO3 (100 mL) and 2 M aq HCl (100 mL). The aq layers were re-extracted
with CH2Cl2 (2 × 100 mL) and the combined organic layers were dried and concentrated.
Chromatography of the crude product (1:1 EtOAc-hexanes) gave hemiacetal 8 (1.13 g, 77%, α/β
ratio 3:2) pure as a yellow oil. HRESIMS calcd for C14H20O9S [M+Na]+: 387.0726, found
387.0717.
1H NMR (400 MHz, CDCl3, 297 K): δ 5.52–5.50 (m, 2H, H-1α, H-4α), 5.45–5.44 (m, 1H, H-4β),
5.40 (dd, 1H, J = 3.3, 10.9 Hz, H-2α), 5.16 (dd, 1H, J = 3.4, 10.8 Hz, H-3α), 5.08–5.06 (m, 2H,
H-2β, H-3β), 4.68 (dd, 1H, J = 8.6, 15.2 Hz, H-1β), 4.28 (m, 1H, H-5α), 3.78–3.74 (m, 1H, H-
5β), 3.67 (d, 1H, J = 8.8 Hz, OH-1β), 3.19 (d, 1H, J = 1.1 Hz, OH-1α), 3.16–2.97 (m, 4H, H-6α,
H-6β), 2.36 (s, 6H, α,β-SCOCH3), 2.20, 2.12, 2.01 (3s, 18H, α,β-OCOCH3).
64
13C NMR (100 MHz, CDCl3, 297 K): δ 194.7, 194.6, 171.3, 170.4, 170.3, 170.3, 170.0, 170.0
(α,β-C=O × 8), 95.9 (C-1β), 90.7 (C-1α), 72.3 (C-5β), 71.0 (C-3β), 70.5 (C-2β), 68.9 (C-4α),
68.2 (C-2α or C-3α), 67.9 (C-4β), 67.6 (C-5α), 67.4 (C-2α or C-3α), 30.5, 30.4 (α,β-SCOCH3),
28.4 (C-6α, C-6β), 20.9, 20.7 (α,β-OCOCH3 × 6).
65
5.2.8 The Synthesis of 9: 2,3,4-Tri-O-acetyl-6-S-acetyl-1-trichloroacetimidate-α-D-galactose
a) Cl3CCN, DBU, CH2Cl2
Hemiacetal 8 (1.07 g, 2.94 mmol) was dissolved in anhyd CH2Cl2 (15 mL) under N2 then
trichloroacetonitrile (0.880 mL, 8.81 mmol, 3 equiv) followed by DBU (110 µL, 0.73 mmol,
0.25 equiv) were added and the reaction mixture was stirred for 3 h at rt. The solvent was
evaporated and the residue was purified by chromatography (3:7 EtOAc:hexanes containing
0.1% w/v triethylamine) to give trichloroacetimidate 9 (1.19 g, 2.34 mmol, 79%) pure as a white
foam. [α]D 92° (c 1.0, CH2Cl2), HRESIMS calcd for C16H20O9NSCl3 [M+Na]+: 529.9822, found
529.9819.
1H NMR (400 MHz, CDCl3, 297 K): δ 8.64 (s, 1H, NH), 6.55 (d, 1H, J = 3.4 Hz, H-1), 5.55 (dd,
1H, J = 1.08, 3.0 Hz, H-4), 5.39–5.29 (m, 2H, H-2, H-3), 4.21–4.20 (m, 1H, H-5), 3.09–2.92 (m,
2H, H-6), 2.28 (s, 3H, SCOCH3), 2.16, 2.00, 1.99 (3s, 9H, OCOCH3).
13
C NMR (100 MHz, CDCl3, 298 K): δ 194.3, 170.2, 170.1, 169.9, 160.9 (C=O × 4), 93.4 (C-1),
70.3 (C-4), 68.2 (C-3), 67.8 (C-2), 66.8 (C-5), 30.4 (SCOCH3), 28.4 (C-6), 20.5 (OCOCH3 × 3).
66
5.2.9 The Synthesis of 1156
: 1-Iodododecane
a) PPh3, I2, imidazole
Dodecanol 10 (1.02 g, 5.5 mmol) was dissolved in toluene (140 mL) then PPh3 (5.89 g, 22.5
mmol), I2 (4.31 g, 17.0 mmol) and imidazole (4.31 g, 22.5 mmol) were added and the reaction
was stirred at 110 °C for 2 h. After cooling to rt, satd aq NaHCO3 (140 mL) was introduced and
this mixture was stirred for 5 min. First, I2 was added until the organic phase remained brown,
then, 20% aq Na2S2O3 was added until the reaction mixture became colourless. This biphasic
mixture was transferred to a separatory funnel such that the aq layer could be drained then the
organic layer was washed with H2O (3 × 70 mL), dried and concentrated. Chromatography of the
crude product (1:9 EtOAc-hexanes) gave iodide 11 (1.1066 g, 68%) pure as a yellow oil.
1H NMR (400 MHz, CDCl3, 295 K): δ 3.16 (t, 2H, J = 7.1 Hz, CH2I), 1.81–1.77 (m, 2H,
CH2CH2I), 1.35–1.34 (m, 2H, CH2CH2CH2I), 1.23–1.22 (m, 16H, CH3(CH2)8CH2CH2CH2I),
1.53 (t, 3H, J = 6.7 Hz, CH3).
13
C NMR (100 MHz, CDCl3, 295 K): δ 33.5 (CH2CH2I), 30.4 (CH2CH2CH2I), 31.8, 29.5, 29.5,
29.3, 29.3, 28.5, 22.6 (CH3(CH2)8(CH2)3I), 14.0 (CH3), 7.3 (CH2I).
This product has previously been described in the literature.56
67
5.2.10 The Synthesis of 1256
: 1-Azidododecane
a) NaN3, DMF
To a solution of iodide 11 (1.11 g, 3.8 mmol) in anhyd DMF (50 mL) was added NaN3 (1.46 g,
22.4 mmol). This mixture was stirred for 27 h at 60 °C then the solvent was evaporated and H2O
(50 mL) was added to the residue. The mixture was extracted with Et2O (3 × 100 mL) then the
combined organic layers were washed with H2O (100 mL), dried and concentrated.
Chromatography (2:98 EtOAc-hexanes) gave azido 11 (554 mg, 70%) pure as a colourless oil.
1H NMR (400 MHz, CDCl3, 296 K): δ 3.23 (t, 2H, J = 7.0 Hz, CH2N3), 1.58–1.57 (m, 2H, J =
7.0 Hz, CH2CH2N3), 1.35–1.34 (m, 2H, CH2CH2CH2N3), 1.25–1.24 (m, 16H,
CH3(CH2)8CH2CH2CH2N3), 0.86 (t, 3H, J = 6.8 Hz, CH3).
13
C NMR (100 MHz, CDCl3, 296 K): δ 51.5 (CH2N3), 31.9, 29.6, 29.5, 29.3, 29.2, 28.8, 26.7,
22.6 (CH3(CH2)10CH2N3), 14.1 (CH3).
This product has previously been described in the literature.56
68
5.2.11 The Synthesis of 1448
: 4-O-(2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl)-1,2,3,6-
tetraacetate-α,β-D-glucopyranose
a) Ac2O, pyr.
α-Lactose 13 (10.00 g) was dissolved in acetic anhydride (100 mL) and pyridine (100 mL) and
was stirred at 50 °C for 1.5 h. The reaction was coconcentrated with toluene (3 × 135 mL) and
the residue was dissolved in CH2Cl2 (500 mL) and washed successively with satd aq NaHCO3
(500 mL) and 2 M aq HCl (500 mL). The aq layers were re-extracted with CH2Cl2 (500 mL) and
the combined organic layers were dried and concentrated. Chromatography of the crude product
(1:1 EtOAc-hexanes) gave peracetylated lactose 14 (18.10 g, 96%, α/β ratio 15:1) pure as a white
foam.
1H NMR (400 MHz, CDCl3, 295 K): δ 6.17 (d, 1H, J = 3.6 Hz, H-1α), 5.62 (d, 1H, J = 8.2 Hz,
H-1β), 5.39 (t, 1H, J = 9.7 Hz, H-3α), 5.28 (d, 1H, J = 3.1 Hz, H-4‟α), 5.04 (dd, 1H, J = 7.9, 10.3
Hz, H-2‟α), 4.94 (dd, 1H, J = 7.9, 10.3 Hz, H-2α), 4.91 (dd, 1H, J = 2.4, 3.6 Hz, H-3‟α), 4.60 (d,
1H, J = 8.0 Hz, H-1‟β), 4.54 (d, 1H, J = 7.8 Hz, H-1‟α), 4.36 (d, 1H, J = 10.7 Hz, H-6aα or H-
6a‟α), 4.06–4.05 (m, 3H, H-6αb, H-6b‟α, H-6aα or H-6a‟α), 3.96 (dd, 1H, J = 2.6, 10.1 Hz, H-5α
or H-5‟α), 3.89 (t, 1H, J = 6.6 Hz, H-5α or H-5‟α), 3.79 (t, 1H, J = 9.5 Hz, H-4), 2.11, 2.09,
2.05, 2.00, 1.99, 1.95, 1.89 (16s, 48H, α,β-OCOCH3).
69
13C NMR (100 MHz, CDCl3, 296 K): δ 174.3, 170.5, 170.3, 170.0, 169.9, 169.9, 169.8, 169.5,
169.4, 169.0, 168.9, 168.8, 168.7 (α,β-C=O × 16), 101.5 (C-1‟β), 100.9 (C-1‟α), 91.3 (C-1β),
88.8 (C-1α), 75.6 (C-4α), 72.4 (C-2‟β), 70.8 (C-3‟α), 70.6 (C-5α or C-5‟α), 70.5 (C-5α or C-5‟α),
69.4 (C-2‟α), 69.3 (C-2α), 69.0 (C-3α), 66.7 (C-4‟α), 61.5 (C-6α or C-6‟α), 60.9 (C-6α or C-6‟α),
20.7, 20.7, 20.6, 20.6, 20.5, 20.4, 20.3 (α,β-OCOCH3 × 16).
This product has previously been described in the literature.48
70
5.2.12 The Synthesis of 1548
: 4-O-(2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl)-2,3,6-tri-O-
acetyl-α-D-glucopyranosyl bromide
a) HBr (33% in AcOH), CH2Cl2
Peracetylated lactose 14 (11.30 g, 16.7 mmol) was dissolved in anhyd CH2Cl2 (143 mL) and HBr
(33% in AcOH, 56 mL) under N2 and this mixture was stirred at rt for 40 min. The reaction was
then diluted with CH2Cl2 (500 mL), washed with satd aq NaHCO3 (500 mL) and the aq layer was
re-extracted with CH2Cl2 (2 × 500 mL). The combined organic layers were dried and
concentrated. Re-crystallization was carried out by dissolving the residue in hot Et2O (150 mL),
placing this mixture at –18 °C for 20 min then filtering it. The white crystals were washed with
cold Et2O (200 mL) and dried over high vacuum to give bromide 15 (9.89 g, 85%) pure as a
yellow oil.
1H NMR (400 MHz, CDCl3, 296 K): δ 6.49 (d, 1H, J = 4.0 Hz, H-1), 5.52 (t, 1H, J = 9.6 Hz, H-
3), 5.32 (dd, 1H, J = 0.8, 3.4 Hz, H-4‟), 5.10 (dd, 1H, J = 7.9, 10.4 Hz, H-2‟), 4.92 (dd, 1H, J =
3.4, 10.4 Hz, H-3‟), 4.72 (dd, 1H, J = 4.1, 10.0 Hz, H-2), 4.48 (d, 1H, J = 7.9 Hz, H-1‟), 4.45–
4.44 (m, 1H, H-6a or H-6a‟), 4.19–4.02 (m, 4H, H-5, H-6b, H-6b‟, H-6a or H-6a‟). 3.87–3.84
(m, 2H, H-4, H-5‟), 2.13, 2.10, 2.06, 2.04, 2.03, 2.02 (7s, 21H, OCOCH3).
71
13C NMR (100 MHz, CDCl3, 297 K): δ 170.3, 170.2, 170.1, 170.1, 170.0, 169.2, 168.9 (C=O ×
7), 100.8 (C-1), 86.3 (C-1‟), 74.9 (C-4 or C-5‟), 73.0 (C-5), 71.0 (C-2, C-3‟, C-4 or C-5‟), 69.6
(C-3), 69.0 (C-2‟), 66.6 (C-4‟), 61.0 (C-6 or C-6‟), 60.8 (C-6 or C-6‟), 20.8, 20.7, 20.6 (OCOCH3
× 7).
This product has previously been described in the literature.48
72
5.2.13 The Synthesis of 1649
: 2-Propyn-1-yl 4-O-(2,3,4,6-tetra-O-acetyl-β-D-galactopyranosyl)-
2,3,6-tri-O-acetyl-β-D-glucopyranoside
a) Propargyl alcohol, AgOTf, Ag2CO3, CH2Cl2
Propargyl alcohol (62 µL, 1.07 mmol, 25 equiv), AgOTf (11.1 mg, 0.043 mmol, 1 equiv),
Ag2CO3 (11.8 mg, 0.043 mmol, 1 equiv) and MS 3 Å (80 mg) were stirred in anhyd CH2Cl2 (0.5
mL) under N2 at RT for 30 min. Following this, the mixture was stirred at –10 °C for 5 min then
bromide 15 (0.030 g, 0.0429 mmol, 1 equiv) was added as a solution in CH2Cl2 (0.5 mL). The
reaction was held at –10 °C for 1 h then allowed to warm to rt over the next 18 h. This mixture
was filtered over a pad of Celite® that was washed with CH2Cl2 (10 mL). The filtrate was washed
with NaHCO3 (10 mL) and the aq phase was re-extracted with CH2Cl2 (3 × 10 mL). The
combined organic layers were dried and concentrated. Chromatography of the crude product (6:4
EtOAc-hexanes) gave propargyl lactoside 16 (17.9 mg, 62%) pure as a white foam.
1H NMR (400 MHz, CDCl3, 295 K): δ 5.32 (dd, 1H, J = 0.92, 3.4 Hz, H-4‟), 5.20 (t, 1H, J = 9.3
Hz, H-3), 5.07 (dd, 1H, J = 7.9, 10.4 Hz, H-2‟), 4.94–4.87 (m, 2H, H-2, H-3‟), 4.71 (d, 1H, J =
7.9 Hz, H-1), 4.50–4.49 (m, 1H, H-6a), 4.45 (d, 1H, J = 7.9 Hz, H-1‟), 4.30 (t, 2H, J = 2.4 Hz,
OCH2C≡CH), 4.13–4.03 (m, 3H, H-6b, H-6a‟, H-6b‟), 3.86–3.82 (m, 1H, H-5‟), 3.79 (t, 1H, J =
9.7 Hz, H-4), 3.63–3.61 (m, 1H, H-5), 2.43 (t, 1H, J = 2.4 Hz, C≡CH), 2.12, 2.10, 2.03, 2.03,
2.02, 2.02 (7s, 21H, OCOCH3).
73
13C NMR (100 MHz, CDCl3, 295 K): δ 170.4, 170.2, 170.1, 169.8, 169.1, 101.1 (C-1‟), 97.9 (C-
1), 78.1 (C≡CH), 76.1 (C-4), 75.5 (C≡CH), 72.8 (C-3), 72.7 (C-5), 71.3 (C-2), 71.0 (C-5‟), 70.7
(C-3‟), 69.1 (C-2‟), 66.6 (C-4‟), 61.8 (C-6‟), 60.8 (C-6), 55.9 (OCH2C≡CH), 20.9, 20.8, 20.7,
20.7, 20.5 (OCOCH3 × 5).
This product has previously been described in the literature.49
74
5.2.14 The Synthesis of 1749
: 2-Propyn-1-yl 4-O-(β-D-galactopyranosyl)-β-D-glucopyranoside
a) NaOMe/MeOH
Sodium (375 mg, 16.3 mmol) was added to a solution of disaccharide 16 (5.47 g, 8.1
mmol) in MeOH (82 mL) and the reaction mixture was stirred at rt for 1 h. This mixture was
diluted with MeOH (200 mL), neutralized with Dowex H+ resin, filtered and the resin was
washed with MeOH (200 mL). The filtrate was concentrated and the residue was dried under
high vacuum to give compound 17 (2.81 g, 91%) pure as a yellow oil.
1H NMR (400 MHz, MeOD, 295 K): δ 4.49 (d, 1H, J = 7.8 Hz, H-1), 4.40 (t, 2H, J = 2.5 Hz,
OCH2C≡CH), 4.34 (d, 1H, J = 7.5 Hz, H-1‟), 3.89 (dd, 1H, J = 2.4, 12.2 Hz, H-6), 3.85–3.79
(m, 2H, H-6, H-4‟), 3.79–3.66 (m, 2H, H-6a‟, H-6b‟), 3.59–3.45 (m, 5H, H-2‟, H-3, H-3‟, H-4,
H-5‟), 3.42–3.39 (m, 1H, H-5), 3.26 (t, 1H, J = 8.1 Hz, H-2), 2.86 (t, J = 2.4 Hz, 1H, C≡CH).
13
C NMR (100 MHz, MeOH, 296 K): δ 105.1 (C-1‟), 101.9 (C-1), 80.5 (C-4), 77.1 (C-5‟), 76.6
(C-5), 76.4 (C-2 or C-3‟), 76.3 (C≡CH), 75.9 (C≡CH), 74.8 (C-3‟), 74.5 (C-2), 72.6 (C-2‟ or C-
3), 70.3 (C-4‟), 62.5 (C-6‟), 61.8 (C-6), 56.6 (OCH2C≡CH).
This product has previously been described in the literature.49
75
5.2.15 The Synthesis of 19: 2-Propyn-1-yl 4-O-(2,3-di-O-acetyl-4,6-O-benzylidene-β-D-
galactopyranosyl)-2,3,6-tri-O-acetyl-β-D-glucopyranoside
a) PhCH(OMe)2, CSA, MeCN; b) Ac2O, pyr.
Deprotected propargyl lactoside 17 (2.81 g, 7.39 mmol) was suspended in anhyd acetonitrile
(281 mL) under N2. Benzaldehyde dimethyl acetal (5.01 mL, 33.2 mmol, 4.5 equiv) was added
followed by CSA (1.12 g, 4.82 mmol, 0.65 equiv) and the reaction mixture was stirred at 70 °C
for 2 h. The reaction was quenched with Et3N (0.720 mL, 5.17 mmol, 0.7 equiv) and the solvent
was evaporated. Chromatography of the crude product (5:95 MeOH:CH2Cl2) gave impure
disaccharide 18 (2.14 g). Impure compound 18 (2.14 g) was dissolved in acetic anhydride (16.5
mL) and pyridine (16.5 mL) and the reaction mixture was stirred at 50 °C for 45 min. The
solvent was then co-evaporated with toluene (20 mL). The residue was dissolved in CH2Cl2 (50
mL) and washed successively with satd aq NaHCO3 (50 mL) and 2 M aq HCl (50 mL). The aq
layers were re-extracted with CH2Cl2 (2 × 50 mL) and the combined organic layers were dried
and concentrated. Chromatography of the crude product (1:1 EtOAc-hexanes) gave pure
disaccharide 19 (2.86 g, 57% from 17) pure as a colourless glass. [α]D 22° (c 1.0, CH2Cl2),
HRESIMS calcd for C32H38O16 [M+NH4]+: 696.2504, found 696.2527.
76
1H NMR (400 MHz, CDCl3, 295 K): δ 7.45–7.42 (m, 2H, Ar), 7.36–7.35 (m, 3H, Ar), 5.45 (s,
1H, CH acetal), 5.26–5.20 (m, 2H, H-2‟, H-3), 4.93 (dd, 1H, J = 7.9, 9.7 Hz, H-2), 4.85 (dd, 1H,
J = 3.6, 10.3 Hz, H-3‟), 4.72 (d, 1H, J = 8.0 Hz, H-1), 4.51 (dd, 1H, J = 2.0, 12.0 Hz, H-6a), 4.44
(d, 1H, J = 7.9 Hz, H-1‟), 4.32–4.30 (m, 4H, OCH2C≡CH, H-4‟, H-6a‟), 4.10 (dd, 1H, J = 5.0,
12.1 Hz, H-6b), 4.02 (dd, 1H, J = 1.5, 12.4 Hz, H-6b‟), 3.80 (t, 1H, J = 9.7 Hz, H-4), 3.63–3.59
(m, 1H, H-5), 3.44 (s, 1H, H-5‟), 2.43 (t, 1H, J = 2.4 Hz, C≡CH), 2.10, 2.02, 2.02, 2.01 (5s, 15H,
OCOCH3).
13
C NMR (100 MHz, CDCl3, 296 K): δ 170.8, 170.4, 170.2, 169.8, 168.9 (C=O × 5), 137.4 (Ar
quaternary), 129.2, 128.3, 126.5 (Ar), 101.4 (CH acetal), 101.1 (C-1‟), 98.1 (C-1), 78.1 (C≡CH),
75.9 (C-4), 75.5 (C≡CH), 73.1 (C-4‟), 73.0 (C-5), 72.3 (C-3), 72.1 (C-3‟), 71.1 (C-2), 69.0 (C-
2‟), 68.4 (C-6‟), 66.5 (C-5‟), 61.8 (C-6), 55.9 (OCH2C≡CH), 20.9, 20.9, 20.8, 20.7, 20.7
(OCOCH3 × 5).
77
5.2.16 The Synthesis of 20: 2-Propyn-1-yl 4-O-(2,3-di-O-acetyl-β-D-galactopyranosyl)-2,3,6-
tri-O-acetyl-β-D-glucopyranoside
a) 80% aq AcOH
Disaccharide 19 (30.0 mg, 0.044 mmol) was stirred in 80% aq AcOH (2 mL) at 100 °C for 45
min then the reaction was coconcentrated with toluene (2 × 5 mL). The residue was dissolved in
CH2Cl2 (10 mL) and washed with satd aq NaHCO3 (10 mL). The aq layers were re-extracted
with CH2Cl2 (3 × 10 mL). The organic layers were combined, dried and concentrated.
Chromatography of the crude product (5:95 MeOH-CH2Cl2) gave diol 20 (23.3 mg, 89%) pure as
a colourless glass. [α]D –21° (c 0.5, CH2Cl2), HRESIMS calcd for C25H34O16 [M+NH4]+:
608.2191, found 608.2175.
1H NMR (400 MHz, CDCl3, 297 K): δ 5.22–5.14 (m, 2H, H-3, H-2‟), 4.91–4.83 (m, 2H, H-2, H-
3‟), 4.70 (d, 1H, J = 7.8 Hz, H-1), 4.50–4.46 (m, 2H, H-1‟, H-6a), 4.30 (t, 2H, J = 2.3 Hz,
OCH2C≡CH), 4.10–4.04 (m, 2H, H-6b, H-4‟), 3.89–3.78 (m, 3H, H-4, H-6a‟, H-6b‟), 3.66–3.61
(m, 1H, H-5), 3.55–3.52 (m, 1H, H-5‟), 3.25, 2.75 (bs, 1H, OH-4‟, OH-6‟), 2.43 (t, 1H, J = 2.4
Hz, C≡CH), 2.08, 2.08, 2.05, 2.05, 2.04 (5s, 15H, OCOCH3).
13
C NMR (100 MHz, CDCl3, 297 K): δ 170.5,170.5, 170.2, 169.8, 169.4 (C=O × 5), 101.0 (C-
1‟), 97.8 (C-1), 78.0 (C≡CH), 76.1 (C-4), 75.4 (C≡CH), 74.4 (C-5‟), 73.4 (C-3‟), 73.1 (C-3), 72.7
(C-5), 71.2 (C-2), 69.6 (C-2‟), 67.8 (C-4‟), 62.1 (C-6, C-6‟), 55.8 (OCH2C≡CH), 20.9, 20.8,
20.7, 20.7, 20.6 (OCOCH3 × 5).
78
5.2.17 The Synthesis of 21: 2-Propyn-1-yl 4-O-[6-O-(2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-β-D-
galactopyranosyl)-2,3-di-O-acetyl-β-D-galactopyranosyl]-2,3,6-tri-O-acetyl-β-D-
glucopyranoside
a) AgOTf, CH2Cl2
Bromide 7 (80.9 mg, 0.189 mmol), diol 20 (144 mg, 0.244 mmol, 1.2 equiv) and activated
powdered MS 4 Å (127 mg) were stirred in anhyd CH2Cl2 (8 mL) under N2 for 15 min at 5 °C.
After, AgOTf (37.0 mg, 0.143 mmol, 0.8 equiv) was added. The reaction mixture was allowed to
warm to 10 °C over 45 min then was washed with satd aq NaHCO3 (30 mL). The aq layer was
re-extracted with CH2Cl2 (3 × 30 mL) and the organic layers were combined, dried and
concentrated. Chromatography of the crude product (6:4 EtOAc-hexanes) followed by further
purification by reverse phase HPLC (H2O-CH3CN, 70:30→30:70) gave trisaccharide 21 (97.3
mg, 55%) pure as a white foam. [α]D 45° (c 0.5, CH2Cl2), HRESIMS calcd for C39H51O24S
[M+NH4]+: 954.2913, found 954.2902.
1H NMR (400 MHz, CDCl3, 296 K): δ 5.48 (d, 1H, J = 3.2 Hz, H-4”), 5.20–5.09 (m, 3H, H3,
H2‟, H2”), 4.97 (d, 1H, J = 10.4 Hz, H3”), 4.90–4.83 (m, 2H, H-2, H-3‟), 4.70 (d, 1H, J = 7.9
Hz, H-1), 4.49–4.45 (m, 2H, H-1‟, H-6a), 4.40 (d, 1H, J = 7.8 Hz, H-1”), 4.30 (t, 2H, J = 2.3 Hz,
OCH2C≡CH), 4.10–4.05 (m, 2H, H-4‟, H-6b), 4.00 (dd, 1H, J = 6.7, 10.3 Hz, H-6a‟), 3.79–3.74
(m, 2H, H-6b‟, H-4), 3.70–3.66 (m, 1H, H-5”), 3.61–3.58 (m, 2H, H-5‟, H-5), 3.07–2.96 (m, 2H,
H-6a”, H-6b”), 2.52 (bs, 1H, OH-4”), 2.43 (t, 1H, J = 2.4 Hz, C≡CH), 2.32 (s, 3H, SCOCH3),
2.09, 2.05, 2.05, 2.03, 2.01, 2.01, 2.01, 2.00 (8s, 24H, OCOCH3).
79
13C NMR (100 MHz, CDCl3, 297 K): δ 194.6, 170.5, 170.3, 170.1, 170.0, 169.8, 169.7, 169.6,
169.3 (C=O × 9), 101.0 (C-1‟), 100.9 (C-1”), 97.9 (C-1), 78.1 (C≡CH), 75.9 (C-4), 75.4 (C≡CH),
73.2 (C-3‟, C-5, C-5‟), 72.8 (C-3), 72.4 (C-5”), 71.2 (C-2), 70.9 (C-3”), 69.6 (C-2”), 68.5 (C-2”),
68.0 (C-4”), 67.2 (C-6‟), 66.6 (C-4‟), 61.9 (C-6), 55.9 (OCH2C≡CH), 30.5 (SCOCH3), 28.6 (C-
6”), 20.9, 20.9, 20.8, 20.7, 20.6, 20.5 (OCOCH3 × 8).
80
5.2.18 The Synthesis of 22: 1-Dodecane-4-(4-O-[6-O-(2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-β-D-
galactopyranosyl)-2,3-di-O-acetyl-β-D-galactopyranosyl]-2,3,6-tri-O-acetyl-β-D-
glucopyranosyloxymethyl)-[1,2,3]-triazole
a) CuI, DIPEA, THF
Trisaccharide 21 (20.6 mg, 0.022 mmol) and azide 12 (16.0 mg, 0.076 mmol, 3.4 equiv) were
dissolved in anhyd THF (1.5 mL) under N2 then CuI (1.6 mg, 0.008 mmol, 0.38 equiv) was
introduced followed by DIPEA (10.0 µL, 0.057 mmol, 2.6 equiv). The reaction mixture was
stirred at 25 °C for 20 h then the solvent was evaporated. The residue was dissolved in CH2Cl2
(10 mL) and washed successively with satd aq NH4Cl (2 × 10 mL) and satd aq NaHCO3 (2 × 10
mL). The aq layer was re-extracted with CH2Cl2 (4 × 10 mL) and the organic layers were
combined, dried and concentrated. Chromatography of the crude product (7:3 EtOAc-hexanes)
gave triazole 22 (14.9 mg, 59%) pure as a white foam. [α]D 113° (c 0.5, CH2Cl2), HRESIMS
calcd for C51H76O24SN3 [M+H]+: 1148.47, found 1148.466.
81
1H NMR (400 MHz, CDCl3, 295 K): δ 7.47 (s, 1H, =CH), 5.38 (d, 1H, J = 2.7 Hz, H-4”), 5.17–
5.10 (m, 3H, H-3, H-2‟, H-2”), 4.98 (d, 1H, J = 10.4 Hz, H-3”), 4.91–4.84 (m, 3H, H-2, H-3‟,
OCHaHb), 4.77 (d, 1H, J = 12.6 Hz, OCHaHb), 4.60 (d, 1H, J = 7.9 Hz, H-1), 4.50–4.47 (m, 2H,
H-1‟, H-6a), 4.42 (d, 1H, J = 7.9 Hz, H-1”), 4.31 (t, 2H, J = 7.2 Hz, NCH2(CH2)10CH3), 4.11–
4.07 (m, 2H, H-6b, H-4‟), 4.00 (dd, 1H, J = 6.8, 10.3 Hz, H-6a‟), 3.79–3.74 (m, 2H, H-4, H-6b‟),
3.71–3.67 (m, 1H, H-5”), 3.62–3.59 (m, 2H, H-5, H-5‟), 3.08–2.97 (m, 2H, H-6a”, H-6b”), 2.38
(bs, 1H, OH-4”), 2.32 (s, 3H, SCOCH3), 2.16, 2.10, 2.06, 2.05, 2.03, 2.02, 1.96, 1.95 (8s, 24H,
OCOCH3), 1.86 (t, 2H, J = 7.3 Hz, NCH2CH2(CH2)9CH3), 1.29–1.23 (m, 18H,
NCH2CH2(CH2)9CH3), 0.85 (t, 3H, J = 6.7 Hz, N(CH2)11CH3).
13
C NMR (100 MHz, CDCl3, 297 K): δ 194.7, 170.1, 169.9, 169.2 (C=O × 9), 144.1(C=CH),
122.3 (C=CH), 101.0 (C-1‟), 100.8 (C-1”), 99.7 (C-1), 75.8 (C-4), 73.2 (C-3‟), 73.1 (C-5, C-5‟),
72.8 (C-3), 72.3 (C-5”), 71.5 (C-2), 70.8 (C-3”), 69.6 (C-2‟), 68.6 (C-2”), 68.0 (C-4”), 67.2 (C-
6‟), 66.6 (C-4‟), 63.1 (OCH2), 61.9 (C-6), 50.4 (NCH2(CH2)10CH3), 31.9 (NCH2CH2(CH2)9CH3),
30.5 (SCOCH3), 30.3 (NCH2CH2(CH2)9CH3), 29.6, 29.5, 29.3, 29.3, 29.0
(NCH2CH2(CH2)9CH3), 28.6 (C-6”), 26.5, 22.6 (NCH2CH2(CH2)9CH3), 20.9, 20.8, 20.7, 20.6
(OCOCH3 × 8), 14.1 (N(CH2)11CH3).
82
5.2.19 The Synthesis of 23: 1-Dodecane-4-(4-O-[6-O-(6-thio-β-D-galactopyranosyl)-β-D-
galactopyranosyl]-β-D-glucopyranosyloxymethyl)-[1,2,3]-triazole disulfide
a) NaOMe/MeOH
Sodium (3.3 mg, 0.14 mmol) was added to a solution of trisaccharide 22 (63.6 mg, 0.055 mmol)
in anhyd MeOH (1.7 mL) and the reaction mixture was stirred at rt under N2. After 40 min, the
reaction mixture was quenched with 20% methanolic AcOH until pH strip indicated neutral. The
solvent was evaporated and the residue was dissolved in methanol and stirred at 40 °C for 20 h.
The solvent was again evaporated and chromatography of the residue (5:95 MeOH-CH2Cl2 →
4:6 MeOH-CH2Cl2) gave disulfide 23 (32.2 mg, 75%) pure as a white foam. [α]D 14° (c 0.5,
MeOH), HRESIMS calcd for C66H116O30S2N6 [M+Na]+: 1559.7075, found 1559.7059.
1H NMR (400 MHz, CD3OD, 296 K): δ 8.00 (s, 2H, =CH), 4.95 (d, 2H, J = 12.4 Hz, OCHaHb),
4.77 (d, 2H, J = 12.4 Hz, OCHaHb), 4.43–4.35 (m, 8H, NCH2(CH2)10CH3, H-1, H-1‟), 4.31 (d,
2H, J = 7.4 Hz, H-1”), 4.03–3.80 (m, 14H, H-4”, H-5, H-5‟, H-6a, H-6b, H-6a‟, H-6b‟), 3.77–
3.74 (m, 2H, H-5”), 3.59–3.47 (m, 14H, H-2 or H-2‟, H-2”, H-3, H-3‟, H-3”, H-4, H-4‟), 3.29–
3.28 (m, 2H, H-2 or H-2‟), 3.03–3.00 (m, 4H, H-6a”, H-6b”), 1.89 (t, 4H, J = 7.0 Hz,
NCH2CH2(CH2)9CH3), 1.32–1.28 (m, 36H, NCH2CH2(CH2)9CH3), 0.88 (t, 6H, J = 6.6 Hz,
N(CH2)11CH3).
83
13C NMR (150 MHz, CD3OD, 295 K): δ 145.7 (C=CH), 125.3 (C=CH), 105.5, 105.1 (C-1 or C-
1‟, C-1”) 103.3 (C-1 or C-1‟), 82.5, 76.6, 76.3, 75.8, 74.8, 74.6, 74.6, 74.5, 72.6, 72.4, 72.4, 71.4,
70.4, 70.2 (C-2, C-2‟, C-2”, C-3, C-3‟, C-3”, C-4, C-4‟, C-4”, C-5, C-5‟, C-5”), 63.1 (OCH2),
62.2 (C-6, C-6‟), 51.4 (NCH2(CH2)10CH3), 41.2 (C-6”), 31.4 (NCH2CH2(CH2)9CH3), 33.1, 30.8,
30.7, 30.6, 30.5, 30.2, 27.5, 23.8 (NCH2CH2(CH2)9CH3), 14.5 (N(CH2)11CH3).
84
5.2.20 Synthesis of 24: 2-Propyn-1-yl 4-O-(2,3,6-tri-O-acetyl-β-D-galactopyranosyl)-2,3,6-tri-
O-acetyl-β-D-glucopyranoside
a) AcCl, collidine, CH2Cl2
Diol 20 (50.0 mg, 0.085 mmol) and collidine (169 µL, 1.27 mmol, 15.0 equiv) were stirred in
anhyd CH2Cl2 (2 mL) under N2 at –35 °C for 10 min. AcCl (18 µL, 0.25 mmol, 3.0 equiv) was
introduced and the reaction mixture was held at –35 °C for 1 h then allowed to warm to –5 °C
over the second h. The addition of MeOH (1.5 mL) was followed by co-evaporation of the
solvents with toluene (2 × 2 mL). The residue was dissolved in CH2Cl2 (5 mL) and washed
successively with satd aq NaHCO3 (5 mL) and 2 M aq HCl (5 mL). The aq layers were re-
extracted with CH2Cl2 (2 × 5 mL) and the combined organic layers were dried and concentrated.
Chromatography of the crude product (6:4 EtOAc-hexanes) gave pure disaccharide 24 (37.8 mg,
71%) pure as a colourless glass. [α]D –22° (c 0.5, CH2Cl2), HRESIMS calcd for C27H36O17
[M+NH4]+: 650.2296, found 650.2308.
1H NMR (400 MHz, CDCl3, 295 K): δ 5.19–5.11 (m, 2H, H-3, H-2‟), 4.90–4.81 (m, 2H, H-2, H-
3‟), 4.69 (d, 1H, J = 7.8 Hz, H-1), 4.46 (dd, 1H, J = 1.8, 12.0 Hz, H-6a), 4.39 (d, 1H, J = 1.8, 7.9
Hz, H-1‟), 4.29 (t, 2H, J = 2.4 Hz, OCH2C≡CH), 4.26–4.18 (m, 1H, H-6a‟), 4.10–4.05 (m, 2H,
H-6b, H-6b‟), 3.97 (s, 1H, H-4‟), 3.75 (t, 1H, J = 9.6 Hz, H-4), 3.69–3.66 (m, 1H, H-5‟), 3.63–
3.59 (m, 1H, H-5), 2.82 (s, 1H, OH-4‟), 2.42 (t, 1H, J = 2.4 Hz, C≡CH), 2.08, 2.06, 2.03, 2.01,
2.00 (6s, 18H, OCOCH3).
85
13C NMR (100 MHz, CDCl3, 297 K): δ 170.7, 169.1 (C=O × 6), 100.8 (C-1‟), 97.8 (C-1), 78.0
(C≡CH), 76.0 (C-4), 75.3 (C≡CH), 73.1 (C-3‟), 72.7 (C-5), 72.4 (C-3), 71.9 (C-5‟), 71.1 (C-2),
69.3 (C-2‟), 66.6 (C-4‟), 61.8 (C-6, C-6‟), 55.7 (OCH2C≡CH), 20.7, 20.7, 20.6, 20.5 (OCOCH3 ×
6).
86
5.2.21 The Synthesis of 25: 2-Propyn-1-yl 4-O-(2,3,6-tri-O-acetyl-4-O-
[(trifluoromethyl)sulfonyl]-β-D-galactopyranosyl)-2,3,6-tri-O-acetyl-β-D-glucopyranoside
a) Tf2O, pyr., CH2Cl2
Disaccharide 24 (66.2 mg, 0.11 mmol) was dissolved in anhyd CH2Cl2 (0.5 mL) and anhyd
pyridine (59 µL, 0.73 mmol, 7 equiv) under N2 and this mixture was cooled to –20 °C. Triflic
anhydride (35 µL, 0.21 mmol, 2 equiv) was added dropwise and the reaction mixture was stirred
at rt for 30 min. CH2Cl2 (15 mL) was added to the mixture that was then washed successively
with 2 M aq HCl (15 mL) and satd aq NaHCO3 (15 mL). The aq layers were re-extracted with
CH2Cl2 (2 × 15 mL) and the combined organic layers were dried and concentrated to give triflate
25 (55.3 mg, 69%) pure as a white foam. [α]D –27° (c 1.0, CH2Cl2), HRESIMS calcd for
C28H35O19SF3 [M+NH4]+: 782.1789, found 782.1824.
1H NMR (400 MHz, CDCl3, 295 K): δ 5.21–5.16 (m, 2H, H-3, H-4‟), 5.11 (dd, 1H, J = 7.7, 10.4
Hz, H-2‟), 5.05 (dd, 1H, J = 3.0, 10.4 Hz, H-3‟), 4.89 (dd, 1H, J = 7.9, 9.4 Hz, H-2), 4.71 (d, 1H,
J = 7.9 Hz, H-1), 4.51 (d, 1H, 7.6 Hz, H-1‟), 4.46 (dd, 1H, J = 2.04, 12.0 Hz, H-6a), 4.35 (dd,
1H, J = 3.5, 8.8 Hz, H-6a‟), 4.30 (t, 2H, J = 2.4 Hz, OCH2C≡CH), 4.06 (dd, 1H, J = 4.8, 12.0 Hz,
H-6b), 3.98–3.91 (m, 2H, H-5‟, H-6b‟), 3.78 (t, 1H, J = 9.6 Hz, H-4), 3.62–3.59 (m, H-5), 2.43
(t, 1H, J = 2.4 Hz, C≡CH), 2.10, 2.08, 2.07, 2.03, 2.03, 2.03, 2.00 (6s, 18H, OCOCH3).
87
13C NMR (100 MHz, CDCl3, 296 K): δ 170.1, 169.8, 169.5, 169.4, 168.4 (C=O × 6), 100.7 (C-
1‟), 97.7 (C-1), 79.8 (C-4‟), 77.8 (C≡CH), 75.9 (C-4), 75.3 (C≡CH), 72.5 (C-5), 72.2 (C-3), 71.0
(C-2), 69.8 (C-5‟), 69.6 (C-3‟), 68.2 (C-2‟), 61.5 (C-6), 60.0 (C-6‟), 55.7 (OCH2C≡CH), 20.6,
20.5, 20.4, 20.3, 20.2 (OCOCH3 × 6).
88
5.2.22 The Synthesis of 26: 2-Propyn-1-yl 4-O-(2,3,6-tri-O-acetyl-β-D-glucopyranosyl)-2,3,6-
tri-O-acetyl-β-D-glucopyranoside
a) NaNO3, DMF
Triflate 25 (55.3 mg, 0.072 mmol) and NaNO3 (61.0 mg, 0.88 mmol) were stirred in anhyd DMF
(4.3 mL) at 50 °C for 20 h under N2. The solvent was then evaporated and the residue was
dissolved in CH2Cl2 (10 mL) and washed with brine (10 mL). The aq layer was re-extracted with
CH2Cl2 (2 × 10 mL) and the combined organic layers were dried and concentrated.
Chromatography of the crude product (6:4 EtOAc-hexanes) gave propargyl cellobioside 26 (23.8
mg, 52%) pure as a white foam. [α]D –53° (c 0.5, CH2Cl2), HRESIMS calcd for C27H36O17
[M+NH4]+: 650.2296, found 650.2292.
1H NMR (400 MHz, CDCl3, 295 K): δ 5.17 (t, 1H, J = 9.4 Hz, H-3), 4.99–4.80 (m, 3H, H-2, H-
2‟, H-3‟), 4.70 (d, 1H, J = 7.9 Hz, H-1), 4.58–4.49 (m, 2H, H-6a, H-6a‟), 4.44 (d, 1H, J = 7.9 Hz,
H-1‟), 4.30 (t, 2H, J = 2.4 Hz, OCH2C≡CH), 4.18 (dd, 1H, J = 1.9, 12.5 Hz, H-6b‟), 4.12–4.05
(m, 1H, H-6b), 3.76 (t, 1H, J = 9.6 Hz, H-4), 3.60–3.56 (m, 1H, H-5), 3.50–3.41 (m, 2H, H-4‟,
H-5‟), 3.17 (bs, 1H, OH-4‟), 2.42 (t, 1H, J = 2.4 Hz, C≡CH), 2.11, 2.10, 2.03, 2.02, 2.01 (5s,
18H, OCOCH3).
13
C NMR (100 MHz, CDCl3, 296 K): δ 171.9, 171.2, 170.3, 169.9, 169.8, 169.3 (C=O × 6),
100.9 (C-1‟), 97.9 (C-1), 78.1 (C≡CH), 76.4 (C-4), 75.5 (C≡CH), 75.1 (C-3‟), 74.3 (C-5‟), 72.9
(C-5), 72.5 (C-3), 71.5 (C-2‟), 71.2 (C-2), 68.2 (C-4‟), 62.6 (C-6‟), 61.7 (C-6), 55.9
(OCH2C≡CH), 20.9, 20.8, 20.7, 20.6, 20.6 (OCOCH3 × 6).
89
5.2.23 The Synthesis of 27: 2-Propyn-1-yl 4-O-[4-O-(2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-β-D-
galactopyranosyl)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl]-2,3,6-tri-O-acetyl-β-D-
glucopyranoside
a) BF3∙Et2O, CH2Cl2
Trichloroacetimidate 9 (305 mg, 0.60 mmol, 4 equiv) and propargyl cellobioside 26 (94.6 mg,
0.12 mmol, 1 equiv) were dissolved in anhyd CH2Cl2 (3.7 mL) under N2 and stirred at 40°C.
BF3∙Et2O (0.029 mL, 0.18 mmol, 1.5 equiv) was added and the reaction mixture was stirred at 40
°C for 1.5 h. The reaction was quenched with Et3N (0.045 mL) and the mixture was diluted with
CH2Cl2 (10 mL) then washed with satd aq NaHCO3 (10 mL). The aq layers were re-extracted
with CH2Cl2 (3 × 10 mL) and the combined organic layers were dried and concentrated to give
trisaccharide 27 (97.5 mg, 67%) pure as a colourless glass. [α]D –10° (c 1.0, CH2Cl2), HRESIMS
calcd for C41H53O25S [M+NH4]+: 996.3019, found 996.2994.
1H NMR (400 MHz, CDCl3, 295 K): δ 5.34 (d, 1H, J = 3.60 Hz, H-4”), 5.20–5.11 (m, 2H, H-3,
H-3‟), 5.03 (dd, 1H, J = 7.8, 10.4 Hz, H-2”), 4.91–4.82 (m, 3H, H-2, H-2‟, H-3”), 4.69 (d, 1H, J
= 7.92 Hz, H-1), 4.52 (dd, 1H, J = 1.8, 12.0 Hz, H-6a), 4.47 (d, 1H, J = 7.9 Hz, H-1‟), 4.33 (d,
1H, J = 7.8 Hz, H-1”), 4.32–4.30 (m, 3H, H-6a‟, OCH2C≡CH), 4.10–4.05 (m, 2H, H-6b, H-6b‟),
3.80–3.73 (m, 2H, H-4, H-4‟), 3.61–3.56 (m, 3H, H-5, H-5‟, H-5”), 3.00–2.98 (m, 2H, H-6a”, H-
6b”), 2.42 (t, 1H, J = 2.3 Hz, C≡CH), 2.33 (s, 3H, SCOCH3), 2.13, 2.11, 2.11, 2.04, 2.02, 2.01,
1.96, 1.92 (8s, 27H, OCOCH3).
90
13C NMR (100 MHz, CDCl3, 297 K): δ 194.3, 170.1, 170.0, 169.8, 169.6, 169.6, 169.1 (C=O ×
10), 100.5 (C-1”), 100.3 (C-1‟), 97.7 (C-1), 77.9 (C≡CH), 76.1 (C-4, C-4‟), 75.2 (C≡CH), 72.6,
72.5 (C-3, C-3‟), 72.7, 72.1, 72.1 (C-5, C-5‟, C-5”), 71.7 (C-2‟), 71.0 (C-2), 70.9 (C-3”), 68.8
(C-2”), 67.1 (C-4”), 62.0 (C-6‟), 61.4 (C-6), 55.6 (OCH2C≡CH), 30.3 (SCOCH3), 28.0 (C-6”),
20.7, 20.6, 20.5, 20.4, 20.3, 20.3 (OCOCH3 × 9).
91
5.2.24 The Synthesis of 28: 1-Dodecane-4-(4-O-[4-O-(2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-β-D-
galactopyranosyl)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl]-2,3,6-tri-O-acetyl-β-D-
glucopyranosyloxymethyl)-[1,2,3]-triazole
a) 1-Azidododecane, CuI, DIPEA, THF
Trisaccharide (26.0 mg, 0.027 mmol) 27 and azide 12 (19.0 mg, 0.090 mmol, 3.4 equiv) were
dissolved in anhyd THF (1.3 mL) under N2 then CuI (2.3 mg, 0.012 mmol, 0.45 equiv) was
introduced followed by DIPEA (12.0 µL, 0.069 mmol, 2.6 equiv). The reaction mixture was
stirred at 26 °C for 18 h then the solvent was evaporated. The residue was dissolved in CH2Cl2
(10 mL) and washed successively with satd aq NH4Cl (2 × 10 mL) and satd aq NaHCO3 (2 × 10
mL). The aq layer was re-extracted with CH2Cl2 (4 × 10 mL) and the organic layers were
combined, dried and concentrated. Chromatography of the crude product (7:3 EtOAc-hexanes)
gave triazole 28 (21.7 mg, 71%) pure as a white foam. [α]D 7° (c 1.0, CH2Cl2), HRESIMS calcd
for C53H78O25SN3 [M+H]+: 1190.48, found 1190.477.
92
1H NMR (400 MHz, CDCl3, 295 K): δ 7.46 (s, 1H, =CH), 5.34 (d, 1H, J = 3.3 Hz, H-4”), 5.15–
5.11 (m, 2H, H-3, H-3‟), 5.03 (dd, 1H, J = 7.9, 10.4 Hz, H-2”), 4.91–4.84 (m, 4H, H-2, H-2‟, H-
3”, OCHaHb), 4.78 (d, 1H, J = 12.8 Hz, OCHaHb), 4.59 (d, 1H, J = 7.9 Hz, H-1), 4.53 (dd, 1H, J
= 0.1, 10.4 Hz, H-6a), 4.47 (d, 1H, J = 7.8 Hz, H-1‟), 4.36 (d, 1H, J = 7.9 Hz, H-1”), 4.32–4.29
(m, 2H, H-6a‟, NCH2(CH2)10CH3), 4.10–4.05 (m, 2H, H-6b, H-6b‟), 3.80–3.72 (m, 2H, H-4, H-
4‟), 3.61–3.57 (m, 3H, H-5, H-5‟, H-5”), 3.00–2.98 (m, 2H, H-6a”, H-6b”), 2.33 (s, 3H,
SCOCH3), 2.11, 2.09, 2.08, 2.01, 1.99, 1.93, 1.90 (9s, 27H, OCOCH3), 1.87 (t, 2H, J = 7.0 Hz,
NCH2CH2(CH2)9CH3), 1.27–1.20 (m, 18H, NCH2CH2(CH2)9CH3), 0.83 (t, 3H, J = 6.5 Hz,
N(CH2)11CH3).
13
C NMR (100 MHz, CDCl3, 296 K): δ 194.4, 170.3, 170.3, 170.2, 170.1, 169.9, 169.7, 169.6,
169.3, 169.1 (C=O × 9), 144.0 (C=CH), 122.5 (C=CH), 100.7 (C-1”), 100.5 (C-1‟), 99.8 (C-1),
76.3, 75.4 (C-4, C-4‟), 72.8, 72.7 (C-3, C-3‟), 72.9, 72.4, 72.3 (C-5, C-5‟, C-5”), 71.9, 71.5, 71.1
(C-2, C-2‟, C-3”), 69.1 (C-2”), 67.3 (C-4”), 63.1 (OCH2), 62.2 (C-6‟), 61.6 (C-6), 50.4
(NCH2(CH2)10CH3), 31.9 (NCH2CH2(CH2)9CH3), 30.5 (SCOCH3), 30.3 (NCH2CH2(CH2)9CH3),
29.6, 29.5, 29.4, 29.3, 29.0 (NCH2CH2(CH2)9CH3), 28.2 (C-6”), 26.5, 22.7
(NCH2CH2(CH2)9CH3), 20.9, 20.8, 20.7, 20.7 (OCOCH3 × 8), 14.1 (N(CH2)11CH3).
93
5.2.25 The Synthesis of 29: 1-Dodecane-4-(4-O-[4-O-(6-thio-β-D-galactopyranosyl)-β-D-
glucopyranosyl]-β-D-glucopyranosyloxymethyl)-[1,2,3]-triazole disulfide
a) NaOMe/MeOH
Trisaccharide 28 (20.6 mg, 0.017 mmol) was dissolved in anhyd MeOH (3.8 mL) and stirred at
40 °C under N2. A 1 M NaOMe/MeOH solution was prepared and 945 µL of this was
introduced. The reaction mixture was stirred at 40 °C for 15 h. The reaction was quenched with
20% methanolic AcOH until pH strip indicated neutral. The solvent was evaporated and the
residue was dissolved in methanol and stirred at 40 °C for 20 h. The solvent was again
evaporated and two successive column chromatographies of the residue (5:95 MeOH-CH2Cl2 →
6:4 MeOH-CH2Cl2) gave disulfide 29 (7.3 mg, 70%) pure as a white foam. [α]D 2° (c 1.0,
MeOH), HRESIMS calcd for C66H116O30S2N6 [M+NH4]+: 1554.7521, found 1554.7538.
1H NMR (400 MHz, DMSO-d6, 295 K): δ 8.10 (s, 2H, =CH), 5.45 (d, 2H, J = 4.9 Hz, OH-2 or
OH-2‟), 5.19–5.18 (m, 4H, OH-2 or OH-2‟, OH), 4.90–4.78 (m, 6H, OCHaHb, OH × 2), 4.68–
4.60 (m, 8H, OCHaHb, OH × 3), 4.46 (bs, 2H, OH), 4.34–4.29 (m, 8H, NCH2(CH2)10CH3, H-1,
H-1‟), 4.27 (d, 2H, J = 7.4 Hz, H-1”), 3.80–3.77 (m, 4H, H-6a, H-6a‟) 3.73–3.69 (m, 2H, H-5”),
3.65–3.56 (m, 6H, H-4”, H-6b, H-6b‟), 3.33–3.32 (m, 16H, H-2”, H-3, H-3‟, H-3”, H-4, H-4‟, H-
5, H-5‟), 3.10–3.04 (m, 4H, H-2, H-2‟), 2.98–2.91 (m, 4H, H-6a”, H-6b”), 1.78 (t, 4H, J = 7.1
Hz, NCH2CH2(CH2)9CH3), 1.22–1.21 (m, 36H, NCH2CH2(CH2)9CH3), 0.84 (t, 6H, J = 6.6 Hz,
N(CH2)11CH3).
94
13C NMR (150 MHz, DMSO-d6, 295 K): δ 143.6 (C=CH), 124.0 (C=CH), 103.7 (C-1”), 102.7,
101.8 (C-1, C-1‟), 80.4, 79.8, 74.9, 74.8, 74.7, 73.1, 72.9, 70.1, (C-2, C-2‟, C-2”, C-3, C-3‟, C-
3”, C-4, C-4‟, C-5, C-5‟, C-5”), 69.1 (C-4”), 61.7 (OCH2), 60.3, 60.2 (C-6, C-6‟), 49.3
(NCH2(CH2)10CH3), 39.4 (C-6”), 29.7 (NCH2CH2(CH2)9CH3), 31.3, 29.0, 29.0, 28.9, 28.7, 28.4,
25.9, 22.1 (NCH2CH2(CH2)9CH3), 14.0 (N(CH2)11CH3).
95
5.2.26 The Synthesis of 3157
: 1-Iodooctadecane
a) PPh3, I2, imidazole
Octadecanol 30 (1.00 g, 3.70 mmol) was dissolved in toluene (199 mL) then PPh3 (3.90 g, 14.8
mmol), I2 (2.80 g, 11.1 mmol) and imidazole (1.00 g, 14.8 mmol) were added and the reaction
was stirred at 110 °C for 2 h. After cooling to rt, satd aq NaHCO3 (100 mL) was introduced and
this mixture was stirred for 5 min. First, I2 was added until the organic phase remained brown,
then, 20% aq Na2S2O3 was added until the reaction mixture became colourless. This biphasic
mixture was transferred to a separatory funnel such that the aq layer could be drained then the
organic layer was washed with H2O (3 × 80 mL), dried and concentrated. Chromatography of the
crude product (2:98 EtOAc-hexanes) gave iodide 31 (1.22 g, 87%) pure as a yellow oil.
1H NMR (400 MHz, CDCl3, 295 K): δ 3.16 (t, 2H, J = 7.1 Hz, CH2I), 1.81–1.78 (m, 2H,
CH2CH2I), 1.37–1.36 (m, 2H, CH2CH2CH2I), 1.23–1.22 (m, 28H, CH3(CH2)14CH2CH2CH2I),
0.86 (t, 3H, J = 6.6 Hz, CH3).
13
C NMR (100 MHz, CDCl3, 296 K): δ 33.6, (CH2CH2I), 30.5 (CH2CH2CH2I), 32.0, 29.7, 29.7,
29.6, 29.6, 29.4, 29.4, 28.6, 22.7 (CH3(CH2)14(CH2)3I), 14.1 (CH3), 7.4 (CH2I).
This product has previously been described in the literature.57
96
5.2.27 The Synthesis of 3257
: 1-Azidooctadecane
a) NaN3, DMF
To a solution of iodide 31 (1.19 g, 3.13 mmol) in anhyd DMF (54 mL) was added NaN3 (1.22 g,
18.8 mmol). This mixture was stirred for 27 h at 60 °C then the solvent was evaporated and H2O
(70 mL) was added to the residue. The mixture was extracted with Et2O (3 × 120 mL) then the
combined organic layers were washed with H2O (120 mL), dried and concentrated.
Chromatography (2:98 EtOAc-hexanes) gave azido 32 (0.86 g, 93%) pure as a colourless oil.
1H NMR (400 MHz, CDCl3, 295 K): δ 3.23 (t, 2H, J = 7.0 Hz, CH2N3), 1.58–1.57 (m, 2H, J =
6.8 Hz, CH2CH2N3), 1.37–1.20 (m, 30H, CH3(CH2)15CH2CH2N3, 0.86 (t, 3H, J = 6.8 Hz, CH3).
13
C NMR (100 MHz, CDCl3, 296 K): δ 51.5 (CH2N3), 31.9, 29.7, 29.6, 29.5, 29.4, 29.2, 28.8,
26.7, 22.7 (CH3(CH2)16CH2N3), 14.1 (CH3).
This product has previously been described in the literature.5
97
5.2.28 The Synthesis of 33: 1-Octadecane-4-(4-O-[4-O-(2,3,4-tri-O-acetyl-6-S-acetyl-6-thio-β-
D-galactopyranosyl)-2,3,6-tri-O-acetyl-β-D-glucopyranosyl]-2,3,6-tri-O-acetyl-β-D-
glucopyranosyloxymethyl)-[1,2,3]-triazole
a) 1-Azidooctadecane, CuI, DIPEA, THF
Trisaccharide (40.0 mg, 0.041 mmol) 27 and azide 32 (41.0 mg, 0.139 mmol, 3.4 equiv) were
dissolved in anhyd THF (2 mL) under N2 then CuI (3.0 mg, 0.016 mmol, 0.38 equiv) was
introduced followed by DIPEA (19.0 µL, 0.106 mmol, 2.6 equiv). The reaction mixture was
stirred at 26 °C for 18 h then the solvent was evaporated. The residue was dissolved in CH2Cl2
(10 mL) and washed successively with satd aq NH4Cl (2 × 10 mL) and satd aq NaHCO3 (2 × 10
mL). The aq layer was re-extracted with CH2Cl2 (4 × 10 mL) and the organic layers were
combined, dried and concentrated. Chromatography of the crude product (6:4 EtOAc-hexanes →
9:1 EtOAc-hexanes) gave triazole 33 (36.4 mg, 70%) pure as a yellow oil. [α]D –6° (c 1.0,
CH2Cl2), HRESIMS calcd for C59H91O25SN3 [M+H]+: 1274.5741, found 1274.5706.
98
1H NMR (400 MHz, CDCl3, 295 K): δ 7.46 (s, 1H, =CH), 5.34 (d, 1H, J = 3.3 Hz, H-4”), 5.15–
5.11 (m, 2H, H-3, H-3‟), 5.03 (dd, 1H, J = 7.9, 10.4 Hz, H-2”), 4.91–4.84 (m, 4H, H-2, H-2‟, H-
3”, OCHaHb), 4.76 (d, 1H, J = 12.6 Hz, OCHaHb), 4.59 (d, 1H, J = 7.9 Hz, H-1), 4.53 (dd, 1H, J
= 1.83, 12.0 Hz, H-6a), 4.47 (d, 1H, J = 7.8 Hz, H-1‟), 4.36 (d, 1H, J = 7.9 Hz, H-1”), 4.32–4.29
(m, 2H, H-6a‟, NCH2(CH2)10CH3), 4.10–4.05 (m, 2H, H-6b, H-6b‟), 3.80–3.72 (m, 2H, H-4, H-
4‟), 3.61–3.57 (m, 3H, H-5, H-5‟, H-5”), 3.00–2.98 (m, 2H, H-6a”, H-6b”), 2.33 (s, 3H,
SCOCH3), 2.11, 2.09, 2.08, 2.05, 2.03, 2.01, 1.99, 1.93, 1.90 (9s, 27H, OCOCH3), 1.87 (t, 2H, J
= 7.0 Hz, NCH2CH2(CH2)9CH3), 1.27–1.20 (m, 30H, NCH2CH2(CH2)15CH3), 0.83 (t, 3H, J = 6.5
Hz, N(CH2)11CH3).
13
C NMR (100 MHz, CDCl3, 296 K): δ 194.4, 170.3, 170.3, 170.2, 170.0, 169.9, 169.7, 169.6,
169.3, 169.1 (C=O × 9), 144.0 (C=CH), 122.5 (C=CH), 100.7 (C-1”), 100.5 (C-1‟), 99.7 (C-1),
76.3, 75.4 (C-4, C-4‟), 72.8, 72.7 (C-3, C-3‟), 72.9, 72.4, 72.3 (C-5, C-5‟, C-5”), 71.9, 71.5, 71.1
(C-2, C-2‟, C-3”), 69.1 (C-2”), 67.3 (C-4”), 63.1 (OCH2), 62.2 (C-6‟), 61.6 (C-6), 50.4
(NCH2(CH2)10CH3), 31.9 (NCH2CH2(CH2)9CH3), 30.5 (SCOCH3), 30.3 (NCH2CH2(CH2)9CH3),
29.7, 29.7, 29.6, 29.5, 29.4, 29.4, 29.0 (NCH2CH2(CH2)9CH3), 28.2 (C-6”), 26.5, 22.7
(NCH2CH2(CH2)9CH3), 20.9, 20.8, 20.7, 20.7 (OCOCH3 × 8), 14.1 (N(CH2)11CH3).
99
5.2.29 The Synthesis of 34: 1-Octadecane-4-(4-O-[4-O-(6-thio-β-D-galactopyranosyl)-β-D-
glucopyranosyl]-β-D-glucopyranosyloxymethyl)-[1,2,3]-triazole disulfide
a) NaOMe/MeOH
Trisaccharide 33 (45.0 mg, 0.035 mmol) was dissolved in anhyd MeOH (890 µL) and stirred at
40 °C under N2. A 1 M NaOMe/MeOH solution was prepared and 223 µL of this was
introduced. The reaction mixture was stirred at 40 °C for 15 h. The reaction was then quenched
with 20% methanolic AcOH until pH strip indicated neutral. The solvent was evaporated and the
residue was dissolved in methanol and stirred at 40 °C for 20 h. The solvent was again
evaporated and the residue was dissolved in CHCl3 (10 mL) and washed with H2O (10 mL). The
aq phase was re-extracted with CHCl3 (5 × 10 mL). The organic layer was concentrated and
chromatography of the residue (1:9 MeOH:CH2Cl2 → 9:1 MeOH:CH2Cl2) gave disulfide 34
(23.0 mg, 75%) pure as a white foam. [α]D 6° (c 1.0, 1:1 MeOH-CH2Cl2), HRESIMS calcd for
C78H140O30S2N6 [M+H]+: 1727.8977, found 1727.9021.
100
1H NMR (400 MHz, DMSO-d6, 295 K): δ 8.11 (s, 2H, =CH), 5.48 (bs, 2H, OH-2 or OH-2‟),
5.20–5.19 (m, 4H, OH-2 or OH-2‟, OH), 4.86–4.83 (m, 4H, OCHaHb, OH-5”), 4.67–4.61 (m,
8H, OCHaHb, OH-6a or OH-6b, OH-6a‟ or OH-6b‟, OH-6a or OH-6b or OH-6a‟ or OH-6b‟),
4.49 (bs, 2H, OH), 4.35–4.30 (m, 8H, NCH2(CH2)16CH3, H-1, H-1‟), 4.26 (d, 2H, J = 7.2 Hz, H-
1”), 3.80–3.78 (m, 4H, H-6a, H-6a‟) 3.74–3.71 (m, 2H, H-5”), 3.66–3.57 (m, 6H, H-4”, H-6b, H-
6b‟), 3.34–3.33 (m, 16H, H-2”, H-3, H-3‟, H-3”, H-4, H-4‟, H-5, H-5‟), 3.10–3.05 (m, 4H, H-2,
H-2‟), 2.98–2.91 (m, 4H, H-6a”, H-6b”), 1.79 (t, 4H, J = 6.8 Hz, NCH2CH2(CH2)15CH3), 1.23–
1.22 (m, 60H, NCH2CH2(CH2)15CH3), 0.85 (t, 6H, J = 6.6 Hz, N(CH2)17CH3).
13
C NMR (150 MHz, DMSO-d6, 295 K): δ 143.6 (C=CH), 124.1 (C=CH), 103.7 (C-1”), 102.8,
101.8 (C-1, C-1‟), 80.4, 79.9, 75.0, 74.8, 74.7, 73.1, 72.9, 70.1 (C-2, C-2‟, C-2”, C-3, C-3‟, C-3”,
C-4, C-4‟, C-5, C-5‟, C-5”), 69.2 (C-4”), 61.7 (OCH2), 60.4, 60.2 (C-6, C-6‟), 49.3
(NCH2(CH2)16CH3), 39.2 (C-6”), 29.8 (NCH2CH2(CH2)15CH3), 31.3, 29.1, 29.0, 28.9, 28.7, 28.4,
25.9, 22.1 (NCH2CH2(CH2)15CH3), 14.0 (N(CH2)17CH3).
101
5.3 Materials and Experimental Methods for Chapter 3
The preparation of the Au(111) electrode used in these studies is described in detail in Dr.
Annia Kycia‟s thesis.21
Before the electrode was used, it was rinsed with Milli-Q water, flame
annealed, allowed to cool then rinsed again and air dried. The area of the Au(111) surface was
0.22 cm2. Glassware was rinsed with Milli-Q ultra pure water then submerged in a solution of
potassium permanganate (4 g KMnO4 per 1 L of Milli-Q water) overnight. Glassware was then
rinsed with a hydrogen peroxide solution (20 mL H2O2 in 250 mL Milli-Q water) followed by
Milli-Q water and dried in an oven at 80 °C.
Passive self-assembly on Au(111) was carried out by immersing the surface of the gold
electrode in a 5 mg/5 mL solution of the analogue overnight (18 to 20 h). The electrode was then
washed with methanol followed by Milli-Q water and argon dried. Samples diluted with 1-β-D-
thioglucose were diluted in a ratio of 80% thioglucose to 20% analogue.
Surface pressure-area isotherms were acquired with a KSV LB Minitrough trough (Total
area 243 cm2). The water phase was maintained at 28 °C for transfer of DMPC/cholesterol and at
20 °C for transfer of the glycolipid analogues. Samples were added to the trough via a
microsyringe and monolayers were left for 15 min to allow for solvent evaporation prior to
compression. Compressions were carried out using a barrier speed of 10 mm/min.
LB transfer to prepare the inner leaflet was carried out by the LB method described in
chapter 1. A 1 mg/mL solution of each analogue in chloroform was prepared and approx. 30 µL
of this was added to the trough until the surface pressure reached 10 mN/m. All of the analogues
were compressed to a surface pressure of 35 mN/m prior to deposition. The dipper rising speed
was set at 20 mm/min to obtain a transfer ratio of 1.0 +/- 0.1. LS touch was performed by the
procedure described in chapter 1 to prepare the outer leaflet. 1,2-Dimyristoyl-sn-glycerol-3-
102
phosphocholine (DMPC) and cholesterol (chol) were purchased from Sigma-Aldrich (99%). The
mixture of DMPC/cholesterol (70:30) was dissolved in chloroform (Fisher, Spectroanalyze
grade) to give a solution of concentration 1.0 mg/mL. Approx. 30 µL of this solution was added
to the trough until a surface pressure of 10 mN/m was reached. Lipids were compressed to a
surface pressure of 35 mN/m prior to LS touch. The same parameters were invoked for LB
transfer of a lipid monolayer that was also described in this thesis.
Vesicle fusion was carried out by adding 2 mL of Milli-Q water to a dried lipid mixture
of DMPC/cholesterol and sonicating this at 40 °C for 1 h. The Au(111) electrode was incubated
in this solution overnight (14 h).
The electrochemical cell was cleaned as described for the glassware. The working
electrode was represented by the Au(111) electrode, the counter electrode was a flame annealed
gold coiled wire and the reference electrode was a saturated calomel electrode (SCE) connected
via a salt bridge. All of the cell potentials in this thesis are quoted versus the SCE scale. The cell
was purged with Argon gas prior to each experiment. The 0.1 M NaF solution (pH = 8.5) used in
all of the electrochemical experiments was prepared by placing NaF powder (VWR Scientific,
Suprapur) in a UV ozone chamber (Jelight, Irvine, CA) for 20 min and dissolving this in Milli-Q
UV-plus ultra pure water (18.2 MΩ∙cm). The electrochemical cell was flushed with argon for 30
min prior to recording measurements. An argon blanket was maintained over the solution
throughout the experiments. The electrode was kept in the hanging-meniscus configuration,
which allows only the single crystal surface to be in contact with the electrolyte.
The stability of every monolayer and bilayer was assessed by cyclic voltammetry
experiments prior to analysis by differential capacitance. The potential was sweeped from 0.45 V
to -0.3 V. CV curves were acquired at a scan rate of 20 mV/s and scan rates that were also tested
103
were 2, 5, 10, 20, 50, 100 and 200 mV/s. The potentiostat was set at a bandwidth of 30 Hz and
the current filter was set at 10 Hz. For the differential capacitance measurements, a scan rate of 5
mV/s was selected and the potentiostat was set at a bandwidth of 100 kHz and the current filter
was set at 100 kHz.
The PM-IRRAS experiments described here were performed with a Nicolet Nexus 870
spectrometer and the details of the apparatus and operation procedures are also described in Dr.
Annia Kycia‟s thesis.21
The gold slides were incubated in 2 mg/mL MeOH solutions for 20 h to
facilitate self-assembly then rinsed with MeOH prior to measurements.
104
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105
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