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Design and Synthesis of
Boronolectin Fluorescence Sensors
Stephan Michael Levonis
BSc (Hons)
The Institute for Glycomics
Griffith University
Submitted in fulfilment of the requirements of the degree of Doctor of
Philosophy
December, 2011
I
Statement of Originality
The content of this thesis has not previously been submitted for a degree or diploma in any
university. To the best of my knowledge and belief, the dissertation contains no material
previously published or written by another person except where due reference is made in
the dissertation itself.
Stephan Michael Levonis BSc (Hons)
II
Preface
Unless otherwise stated, the results in this thesis are those of the author. Parts of this work
have appeared elsewhere.
Included in this thesis are published papers in Chapters 2 and 3 which are co-authored with
other researchers. My contribution to each co-authored paper is outlined at the front of the
relevant chapter. The bibliographic details for these papers are:
Chapter 2:
Levonis, Stephan M.; Kiefel, Milton J.; Houston, Todd A.; Healy, Peter C. 2-Propynyl 2-
hydroxybenzoate. Acta Crystallographica, Section E: Structure Reports Online (2010),
E66(1), o226-o227.
Chapter 3:
Levonis, Stephan M.; Kiefel, Milton J.; Houston, Todd A. Boronolectin with divergent
fluorescent response specific for free sialic acid. Chemical Communications (Cambridge,
United Kingdom) (2009), (17), 2278-2280.
Appropriate acknowledgements of those who contributed to the research but did not
qualify as authors are included in each published paper.
(Signed) _________________________________
Stephan Levonis
IV
Conference Presentations
Oral Presentations
RACI - BBOCS (Brisbane Biological and Organic Chemistry Symposium) November 27, 2009,
Institute for Glycomics, Gold Coast:
“Boronolectins for specific sensing of sialic acid”
American Chemical Society - Spring 2010 National Meeting and Exposition, March 21 - 25,
San Francisco, California:
“Boron acid-catalyzed reactions of -hydroxycarboxylic acids” Levonis, Stephan M.;
Kanesan, Ishvi; Kiefel, Milton J.; Houston, Todd A. Abstracts of Papers, 239th ACS National
Meeting, San Francisco, CA, United States, March 21-25, 2010 (2010), ORGN-76
“Boronolectins for specific sensing of free sialic acid” Levonis, Stephan M.; Kiefel, Milton J.;
Houston, Todd A. Abstracts of Papers, 239th ACS National Meeting, San Francisco, CA,
United States, March 21-25, 2010 (2010), CARB-94.
International Student Forum, September 25 – 29, 2010, Beijing, China:
“Boronolectins for specific sensing of sialic acid”
V
Abbreviations
+ve positive
-ve negative
°C degrees Celsius
B-N Boron-nitrogen
Calcd. Calculated
CD Circular Dichroism
CD3OD d4-methanol
cm-1 wave numbers
CMP Cytidine Monophosphate
DCM dichloromethane
DMF N,N-Dimethylformamide
DMSO-d6 d6-dimethyl sulphoxide
Em. emission wavelength
equiv equivalents
ESIMS ElectroSpray Ionization Mass Spectroscopy
ex. excitation wavelength
VI
FTIR Fourier Transform Infrared Spectroscopy
g gram
H2O water
HPLC High Pressure Liquid Chromatography
HMBC Hetronuclear Multiple Bond Correlation
hr hour
HRMS High Resolution Mass Spectroscopy
H-Bond hydrogen bond
Hz Hertz
IC50 concentration required for 50 % enzyme inhibition
IR Infrared spectroscopy
K2CO3 potassium carbonate
KDN 2-keto-3-deoxy-D-glycero-D-galcto-nononic aicd
KDO keto-3-deoxy-2-octulosonic acid
Ki enzyme binding constant
KOH potassium hydroxide
LPS lipopolysaccharide
VII
m meta
M molarity (mol/L)
MeCN acetonitrile
mg milligram
MHz Megahertz
min minutes
mL milliliter
mmole millimole
mol mole
MS Mass Spectroscopy
nmol nanomoles
NaCl sodium chloride
Na2CO3 sodium carbonate
NaHCO3 sodium bicarbonate
NaOH sodium hydroxide
NMR Nuclear Magnetic Resonance spectroscopy
P para
VIII
PET Photoinduced Electron Transfer
pKa acid dissociation constant (-log Ka)
ppm parts per million
Sialic acid N-acetylneuraminic acid or Neu5Ac
SAR Structure Activity Relationship
t- or tert- tertiary
TLC Thin Layer Chromatography
UV Ultra Violet
g micrograms
L microlitres
mol
micromoles
IX
Acknowledgements
I wish to extend my sincerest thanks to Dr. Todd Houston and Dr. Milton Kiefel. I am very
appreciative of their role as my supervisors and wish to give gratitude for their patience and
advice during my candidature. I feel privileged to have had such excellent supervisors.
Their influence and encouragement has made this task possible and their knowledge has
been invaluable.
I would also like to extend my gratitude to the Institue for Glycomics for support. The
facilities and funding were crucial to the achievement of this work. The environment
provided by the institute was conductive to producing excellent work, with all members of
the institute contributing to provide a supportive and stimulating workplace.
I would like to extend my greatest thanks to my family for their love and support. Mum,
Dad and Ellie.
I would like to thank my fiancée, Stephanie, for her love, understanding, patience, caring
and friendship. Without her support, this work would not have been possible.
X
Table of figures
Tables
Table 1 Acidic carbohydrate esterification using boric acid ................................................ 40
Table 2 Attempted mandelic acid – neutral carbohydrate esterification using boric acid ...... 44 Table 3 Summary of reactions ........................................................................................... 111
Figures
Figure 1 Example of a boronic acid adopting both trigonal and tetrahedral forms in aqueous
environment9 ......................................................................................................................... 3
Figure 2 Depiction of the reversible nature of boronate-diol binding16
.................................. 5
Figure 3 Large dihedral angle at C-O-B-O in the cage shaped borate give less p-orbital
overlap18
............................................................................................................................... 6
Figure 4 Example of carbohydrate binding by boronic acid 24
.............................................. 7
Figure 5 Shinkai’s Photoinduced Electron Transfer based receptor19
.................................. 9
Figure 6 James’s boronic acid based carbohydrate sensors32
.............................................. 10
Figure 7 Illustration of the internal charge transfer effect due to boron’s vacant orbital ..... 12
Figure 8 Bis(boronic acid) fluorescence sensor34,20
............................................................ 12
Figure 9 Sialic acid............................................................................................................ 13 Figure 10 KDO ................................................................................................................ 16
Figure 11 Taylor and Smith’s boronic acid based fluorescence sensor moiety used in a
polymeric sensor65
............................................................................................................... 17
Figure 12 Wang’s “designer” boronolectin ........................................................................ 19 Figure 13 Possible binding modes of a boronic acid to a sialic acid derivative (R = H or Me,
X = OH, N3 or NHC(=NH)NH2)69
....................................................................................... 20
Figure 14 TMR-B bound to the glycerol tail of the galactose aldoxime formed on resin bead 70
......................................................................................................................................... 22
Figure 15 Relative response of the different sugars tested 70
............................................... 22
Figure 16 Mechanistic rationale of boric acid catalysed esterification of an -
hydroxycarboxylic acid81
.................................................................................................... 24
Figure 17 Boric acid catalysed esterification of an -hydroxycarboxylic acid. ................... 28
Figure 18 Potential boronic acid binding sites present on KDO ......................................... 30
Figure 19 Mechanism for the Cornforth method applied to KDO synthesis ....................... 31 Figure 20 Proposed mechanism for boric acid catalysed esterification of sialic acid .......... 42
Figure 21 Proposed complex formed between boric acid, mandelic acid and galactose ....... 43
Figure 22 Slow/Fast mechanism relevant when using equimolar amounts of reactants91
.... 39
Figure 23 View of the two independent molecules in (I) with the atom numbering scheme.
Displacement ellipsoids for non-H atoms are drawn at the 40% probability level. ............... 55 Figure 24 Crystal packing in the structure of (I), viewed down the c axis. .......................... 55
Figure 25 Chart displaying relative fluorescence of imine 52 when combined with different
concentrations of monosaccharides ..................................................................................... 68
Figure 26 Preliminary results showing fluorescence quenching with the addition of sialic
acid in 1:1 methanol/ 100mM aqueous phosphate buffer mixture at pH 6.2 Ex. 295 Em. 365
........................................................................................................................................... 70
XI
Figure 27 ............................................................................................................................. 70 Figure 28 ............................................................................................................................. 71
Figure 29 Fluorescence of m-aminophenylboronic acid in 1:1 MeOH – 200mM aq.
phosphate buffer. (Ex. 287, Em. 400) pH 6.2 ....................................................................... 73
Figure 30 Fluorescence of m-aminophenylboronic acid in 1:1 MeOH – 200mM aq.
phosphate buffer. (Ex. 287, Em. 400) pH 7.8 ....................................................................... 74
Figure 31 Fluorescence of compound 55 in 1:1 MeOH – 200mM aq. phosphate buffer. (Ex.
295, Em. 375). .................................................................................................................... 76
Figure 32 Fluorescence of compound 58 in 1:2 MeOH – 100mM aq. phosphate buffer. (Ex.
275, Em. 375) pH 6.2 .......................................................................................................... 78
Figure 33 Fluorescence of compound 58 in 1:2 MeOH – 100mM aq. phosphate buffer. (Ex.
275, Em. 375) pH 7.8 .......................................................................................................... 79
Figure 34 Boronolectin designed to bind sialic acid. .......................................................... 84
Figure 35 Fluorescence of compound 60 (33 M) in 2 : 1 100 mM aq. phosphate–MeOH
(Ex. 315 nm, Em. 388 nm) pH 7.8 ....................................................................................... 86
Figure 36 Fluorescence of compound 60 (33 M) in 2 : 1 100 mM aq. phosphate–MeOH
(Ex. 315 nm, Em. 388 nm) pH 6.2 ....................................................................................... 87
Figure 37 Possible monosaccharide binding mechanisms of 60 and 58. Only pyranoside
forms shown........................................................................................................................ 88
Figure 38 Fluorescence emmission scan of receptor 62 (Excitiation wavelength = 285 nm)
........................................................................................................................................... 91
Figure 39 Proposed catalytic cycle for amidation using a boronic acid catalyst131
............... 98
Figure 40 1H NMR spectra of crude precipitate from the reaction shown in Scheme 17 ... 101
Figure 41 1H NMR spectrum of 70 (selected regions) ...................................................... 104
Figure 42 13
CNMR spectrum of 70 (selected regions) ...................................................... 105
Figure 43 Possible structures of boron containing heterocycles135
.................................... 109
Figure 44 Proposed structure of compound 70 ................................................................. 110
Figure 45 Proposed structure of compound 67 ................................................................. 110 Figure 46 Example of “on” site activation ....................................................................... 122
Figure 47 Example of “off” site activation ....................................................................... 123 Figure 48 Example of both sites being bound, causing an overall “off” response ............. 124
Figure 49 Mechanistic rationale for the N-alkylation of indoleboronic acid 93................. 125 Figure 50 Alkylation is unlikely to occur at indole position 3 due to solvent choice and ionic
effects ............................................................................................................................... 126 Figure 51 Fluorescence of receptor 92 in 1:2 MeOH – 200 mM aq. phosphate buffer. (Ex.
280, Em. 360) pH 7.0 ........................................................................................................ 129 Figure 52 Fluorescence of receptor 92 in 1:2 MeOH – 200 mM aq. phosphate buffer. (Ex.
280, Em. 360) pH 7.0 ........................................................................................................ 130 Figure 53 Depiction of binding to 5-deoxy KDO producing an “off” response in receptor 92
......................................................................................................................................... 132 Figure 54 Fluorescence of receptor 92 in 1:2 MeOH – 200mM aq. phosphate buffer. (Ex.
280, Em. 360) pH 6.2 ........................................................................................................ 133
XII
Table of Contents
Statement of Originality ......................................................................................................... I Preface ..............................................................................................................................II
Refereed Journal Publications ......................................................................................... III Conference Presentations ................................................................................................ IV
Oral Presentations ........................................................................................................... IV Abbreviations ....................................................................................................................... V
Acknowledgements ......................................................................................................... IX Table of figures ................................................................................................................ X
Chapter 1 .............................................................................................................................. 1 Introduction .......................................................................................................................... 1
1.1 Boron in Nature ........................................................................................................... 2 1.2 The Nature of Boron .................................................................................................... 2
1.3 “Boronolectins” ........................................................................................................... 6 1.4 Specific Target: Sialic Acid ....................................................................................... 13 1.5 Specific Target: KDO ................................................................................................ 15
1.6 “Designer” Boronolectins .......................................................................................... 17 1.7 Catalysis .................................................................................................................... 23
1.8 Project Aims .............................................................................................................. 24 Chapter 2 ............................................................................................................................ 26
Synthesis of Carbohydrate Ligands ..................................................................................... 26 2.1 Overview ................................................................................................................... 27
2.2 Boronolectins, KDO and Sialic Acid ......................................................................... 27 2.3 Project Objectives...................................................................................................... 32
2.4 Preparation of Carbohydrate Derivatives ................................................................... 32 2.4.1 KDO Synthesis ....................................................................................................... 33
2.5 Acta Crystallographica Section E .............................................................................. 47 2.6 Conclusions ............................................................................................................... 56
2.7 Experimental ............................................................................................................. 56 Chapter 3 ............................................................................................................................ 65
The Fluorescence Sensing of Free Sialic Acid ..................................................................... 65 3.1 Free Sialic Acid ......................................................................................................... 66
3.2 Imine Receptor .......................................................................................................... 66 3.2.1 m-Aminophenylboronic acid as a sensor ................................................................. 71
3.2.3 Protection of reactive amine.................................................................................... 74 3.3 Covalently bound receptor ......................................................................................... 76
3.4 Chemical Communications ....................................................................................... 80 3.5 Thiophene ................................................................................................................. 89
3.6 Conclusions ............................................................................................................... 92 3.7 Experimental ............................................................................................................. 92
Chapter 4 ............................................................................................................................ 97 Direct and Rapid Amide Bond Formation ........................................................................... 97
4.1 Amide Coupling ........................................................................................................ 98 4.2 Model Reactions ...................................................................................................... 100
4.2.1 Salicylic acid and o-Aminophenylbornic acid ....................................................... 100 4.2.2 Effects of meta-phenylboronic acid Substitution and Importance of ortho-substitution
...................................................................................................................................... 101 4.2.3 Citric acid and o-Aminophenylboronic acid .......................................................... 102
XIII
4.3 Internal interactions in o-Amidophenylboronic acids ............................................... 105 4.3.1 Proposed Configurations of Reaction Products ..................................................... 109
4.4.1 Mandelic acid and o-Aminophenylboronic acid .................................................... 110 4.4.2 Malic acid and o-Aminophenylboronic acid ........................................................ 110
4.5 Conclusion .............................................................................................................. 113 4.6 Experimental ........................................................................................................... 113
Chapter 5 .......................................................................................................................... 117 Specific Sensing of KDO .................................................................................................. 117
5.1 KDO as the Target ................................................................................................... 118 5.2 Receptor Design ...................................................................................................... 119
5.2.1 Receptor and Substrate Relationship: “On” and “Off” Behaviour.......................... 120 5.2.2 Turning “On” Receptor 92 .................................................................................... 121
5.2.3 Turning “Off” Receptor 92: Single Site Binding ................................................... 123 5.2.4 Turning “Off” Receptor 92: Dual Site Binding ..................................................... 123
5.3 Receptor Synthesis .................................................................................................. 124 5.4 Fluorescence Evaluation of Receptor Performance .................................................. 127
5.5 Binding Sites on KDO ............................................................................................. 131 5.6 Increasing the Acidity: Assay at pH 6.2 ................................................................... 132
5.7 Bidentate Boronic Acids as Esterification Catalysts ................................................. 133 5.8 Conclusions ............................................................................................................. 134
5.9 Experimental ........................................................................................................... 135 Chapter 6 .......................................................................................................................... 138
Conclusions and Future Work ........................................................................................... 138 Appendix .......................................................................................................................... 148
Abstract
This thesis reports on the use of the element boron in organic chemistry. Its role in
catalysis, as well as its broad utility when in the form of a boronic acid functional group is
demonstrated.
Boric acid and boronic acids have applications in numerous kinds of chemical reactions as
catalysts. Boric acid is demonstrated in this work to catalyse the esterification of -
hydroxycarboxylic acid starting materials, including carbohydrates, typically in excellent
yield. A series of reactions were conducted to demonstrate the utility and limitations of this
technique. Included in this work is the synthesis of the carbohydrate, KDO. Furthermore, a
XIV
series of esters were generated using salicylic acid as a starting material, one of which was
subjected to x-ray crystallographic studies.
Also in this thesis a novel type of boronic acid catalysed amide forming reaction is described.
The reaction is shown to proceed rapidly under mild reaction conditions with little
purification required to give a pure product. Structural identification of the amide products
is discussed and hypothesised molecular configurations are presented.
Fluorescence sensors are described as a practical application of boron – polyol interactions.
Supporting theories are outlined and published work is summarised, compared and
contrasted. The carbohydrates sialic acid and KDO are identified as molecular targets for
boronic acid based fluorescence sensors. The benefits of multiple binding sites and
optimised molecular geometry are clearly shown in the results of fluorescence assays.
Sensor molecules reported in this thesis demonstrated selective binding to the
carbohydrates, sialic acid and KDO.
1
Chapter 1
Introduction
2
1.1 Boron in Nature
Boron is an element that is abundant in nature, being present in both plants and
animals, and plays a vital role in the functioning of complex biological systems.1,2 The
element has been shown to be important for the growth of plants by enabling pectic
polysaccharide organisation in leaf cell walls.3 Boron is also considered to be important
for the well being of mammals, including humans, and despite the difficulty in creating
a boron deficient diet it has been demonstrated that in rodents boron is essential for
embryonic development.4 Boron has also been shown to be essential for the health of
fish and amphibians.5-6 It has been shown to affect the behaviour of rats, with boron
deficient rats being less active than rats with an adequate dietary source of boron. The
effect was dependant on the dietary source of fat that the rats consumed, overall the
study demonstrated that boron is an essential trace element for brain health possibly
due to an effect on oxidative metabolism.7 Chemical compounds containing boron
may be toxic or non toxic depending on their structure, most borates have extremely
low toxicity and many grams of a typically available borate would have to be consumed
daily to have deleterious effects in a human.8 Boron is a metalloid element, with
characteristics enabling it to interact with other chemical species in a unique way.
1.2 The Nature of Boron
Boron has the ability to toggle between neutral trigonal planar and tetrahedral forms,
thus boron commonly forms bonds reversibly to oxygen and nitrogen by accepting an
electron pair, as well as retaining any existing covalent bonds.9 The term “boronic
acid” refers to a functional group consisting of boron that is covalently bound to a
carbon, and has two covalently bound hydroxyl groups. When considering the nature
3
of the boron involved in terms of sp2 and sp3 hybridisation, it could be said that there is
present on the element a potentially vacant p orbital available for reversible
interactions, as the boron can adopt both trigonal and tetrahedral binding forms.
Boronic acids rapidly form cyclic esters with diols and polyols in both nonaqueous and
aqueous media at room temperature. Boronic acids form reversible bonds with 1,2- or
1,3-diols including carbohydrates to generate five- or six-membered cyclic complexes.
A boronic acid is routinely incorporated into synthetic receptors designed to complex
compounds possessing 1,2- or 1,3-diol groups. Boronic acids are important organic
intermediates that have been used for applications including the protection of diols,10
Suzuki cross-coupling reactions,11 Diels-Alder reactions,12 and the asymmetric
synthesis of amino acids.13 Such is the interest in this area that a comprehensive
review was published by Wang et al. in 2005 on the topic of boronate binding of
diols/polyols in Medicinal Research Reviews wherein the term “boronolectin” was
used to describe a boronic acid that would bind a particular carbohydrate selectively
over another.14 Another review on the topic of the binding of -hydroxycarboxylic
acids by boronate species was published in 2007 by the Houston group. The former
review outlines previous work conducted on boric acid – diol interactions with an
emphasis on carbohydrate binding. It concludes that there is a need for further
research into the topic of boric acid-diol binding factors to further aid the design of
boronolectins. The 2007 review was concerned primarily with a different kind of
Figure 1 Example of a boronic acid adopting both trigonal and tetrahedral forms in aqueous
environment9
R B
OH
OH
+ 2H2O R B
OH
OH
OH + H3O+
4
interaction, where the binding to -hydroxycarboxylic acid structures was analysed in
depth.15 Advancements in boronic acid chemistry that involve targeting the -
hydroxycarboxylic acid structural motif have appeared only recently in literature. The
history of the reversible nature of boronic acid - diol binding can be traced in literature
to mid last Century, although interactions between boric acid and tartaric acids were
noted in the pioneering work of Jean Baptiste Biot on optical rotation as early as 1832.
In a 1958 study the boronate binding of many diols and polyols including
carbohydrates was reported in detail.16 This study showed a boronic acid will bind to a
diol or polyol covalently, but also reversibly in aqueous solution. It is due to this
behaviour that boronolectins could be developed. The reversible covalent complex
formation was further investigated in subsequent work, which explained that binding
of phenylboronic acid with diols lowered the pKa of phenylboronic acid. In an aqueous
solution, boronic acid can exist in the neutral trigonal planar form 1, or the tetrahedral
anionic form 2, and the preference for one over the other is determined by pH (Figure
2).17 The same is true when bound to a diol like 3, producing the reversible behaviour
as seen in Figure 2. When bound to a diol in neutral aqueous media the boron is
preferentially in the tetrahedral configuration, 5. The general model of equilibrium
holds true for all kinds of aqueous boronic acid diol-type binding behaviour.
5
Figure 2 Depiction of the reversible nature of boronate-diol binding16
In more recent work the pKa of a boronate species was explored by the formation of a
cage-shaped structure using triphenolic methane derivatives to “tune” the geometry
around the boron atom to allow it to become more Lewis acidic (due to favourable
bond angles), and thus become a more effective catalyst. Ab initio calculations
demonstrated in this case that by distorting the geometry around the boron from its
typical planar structure, pi-orbital overlap was reduced and as well as overlap between
p-orbitals on O and B. This work demonstrated that altering the bond angle at the
boron atom will affect its Lewis acidity, as is also true for other metals/metalloids
(Figure 3).18
1 2
3
4 5
B(OH)2 B
HOOH
OHKa-acid
R1
HO
R2
OH
B
O
O
R1
R2
B
O
O
R1
R2
H
R1
HO
R2
OH
Ka-ester
6
Figure 3 Large dihedral angle at C-O-B-O in the cage shaped borate give less p-orbital
overlap18
This work combined with earlier work gives insight into both the reversible nature of
boronic acid-diol binding as well as the changes in Lewis acidity that can occur after
complexation to a covalent ligand. It is these two aspects of boron behaviour that give
the metalloid its desirable behavior when used for the purposes of designing synthetic
lectins.
1.3 “Boronolectins”
Sensors that involve boronic acids have been in use for some time. These include
photoinduced electron transfer (PET) based sensors with one or more binding sites and
polymeric boronic acid-based fluorescence sensors.19,20 There are colorimetric, UV and
CD boronic acid-based sensors currently in use.21,22,23 Another kind of sensor that can
be made by incorporating a boronic acid as an operational feature is a fluorescence
based sensor. The general concept behind this kind of molecular sensor is that since a
boronic acid can bind reversibly to a diol, and when it does so there will be a marked
change in the Lewis acidity of the boron atom, these occurrences can be utilised to
B
O
RO
ORR B
O
RO
OR
R
B
O
O OPh
Ph
PhH
BOOO
2.0o 48.4o
7
generate a compound that will both bind to a diol and exhibit an electronic change
when doing so. This electronic change can alter the fluorescence output of the
molecule indicating the binding event. If the receptor is sufficiently selective for a
particular diol or polyol, this could also be a carbohydrate, it is considered to be a
“boronolectin”. Boronic acid based fluorescence sensors absorb ultra-violet light and
when bound to their substrate will either increase or decrease measurable radiation,
in this way the binding event can be measured.
Figure 4 Example of carbohydrate binding by boronic acid 24
The first boronic acid based sensor for carbohydrates, 2-anthryl boronic acid (6) was
reported by Yoon and Czarnik in 1992 (Figure 4).24 Although it had relatively weak
binding the principle was demonstrated that the formation of the boronate anion
upon esterification to a diol was enough to alter fluorescence. When bound to a
carbohydrate such as fructose the fluorescence output decreased in much the same
way as fluorescent output of compound 6 decreased upon the addition of base. When
base was added to the unbound boronic acid the equilibrium was altered so as to give
the anionic boronate species. It was found that upon binding to fructose a pKa change
from 8.8 to 5.9 was noted and after the complex formed, even in buffered solutions,
the equilibrium was thus pushed towards the anionic boronate species. This caused a
6 7
B
OH
OHB
O
O
O
OH
OH
Fructose
HO
OH
High Fluorescence Low Fluorescence
8
decrease in the observed fluorescent output of the system. This can be due to the fact
that in the trigonal planar configuration this system can be considered to have a
potentially vacant p-orbital on the boron atom. This vacant orbital can effectively
extend the conjugation of the aromatic system by accepting electrons thus enhancing
its fluorescent output. When the boron is coordinated to a diol, the tetrahedral
configuration dominates, eliminating the vacant orbital and reducing the extent of the
conjugated system thus resulting in the lowered fluorescent output. In this work it
was found that if a boronic acid is bound directly to a conjugated fluorescent structure
it will quench fluorescence when a: base is added to raise pH or b: it has bound to a
suitable substrate (usually a diol or polyol). In this way a fluorescence “off” site can be
produced. When considering systems that work in a different way to this, it is notable
that the inclusion of an amine in the structure of boronate carbohydrate sensors can
greatly enhance the fluorescent output through extended conjugation. The first
reported boronic “photoindiced electron transfer” sensor for carbohydrates had a
fluorescence increase so large upon binding that it was labeled an “on-off” type
receptor.19,25 This receptor consisted of a single phenylboronic acid component linked
by an amine to anthracene, to give 10, and displayed quenching behavior in its
unbound form. The unique behavior of this kind of receptor can be explained by
observing the basic theory behind the photoinduced electron transfer process.
Ultra-violet absorbance in organic compounds occurs due to the presence of a
conjugated pi electron system. It is possible for some compounds that use a benzene
fluorophore to have an absorbance maxima as low as 170nm or a high as 300nm.26 It is
not unusual for other reported fluorophores to have an observed maxima of up to
9
600nm. These absorbance maxima correspond to pi pi* transitions as energy is
initially absorbed by the systems. Once the molecule is in an excited electronic state
after being irradiated, it may begin to give up this energy nonradiatively through
collisions with surrounding molecules.27 Eventually it may reach a stage where it is
unable to accept the larger energy difference needed to return to its ground state and
so emits excess energy as radiation. This visible fluorescence will occur at a lower
frequency (longer wavelength) than the incident radiation due to the initial loss of
vibrational energy to the surroundings. There is a mechanism other than collisional
transfer for a molecule in the excited state to give off energy without radiating known
as “charge transfer” and this mechanism is the basis of fluorescence quenching.
Figure 5 Shinkai’s Photoinduced Electron Transfer based receptor19
Photoinduced electron transfer (PET) is a process that can occur in a molecule that has
a quenching component incorporated into its structure and therefore displays a
reduced fluorescence due to electronic effects. PET can be reduced in some cases
when conditions surrounding the quenching group change due to local electronic
8 9
10 11
Strong Fluorescence Quenched Fluorescence
NH NH
Quenched Fluorescence
BB(OH)2
Restored Fluorescence
electron transfer
HO
H
NH2
O
O
10
variations causing fluorescence to be enhanced by a reduction in quenching. If a
naturally fluorescent moiety has a nitrogen atom present with a methylene spacer
from the fluorophore it will experience quenching of fluorescence due to interactions
with nitrogen’s lone pair (Figure 5, 10). So when the system is excited to the pi* state,
as well as collisional transfer of energy, there is also charge transfer as a means of
returning to ground state. If the same molecule contains a proximal boronic acid unit
that can interact with a nitrogen lone pair this will form a simple fluorescence sensor
for diols.28 A diol can potentially complex with the boronic acid subunit causing an
increase of the Lewis acidity of the boron thus increasing boron-nitrogen interactions
(Figure 5, 11). Although the exact nature of the B-N interactions change due to solvent
and pH, the structure 11 shown in Figure 5 is accepted to be correct in an aqueous
environment at around neutral pH.29 By making a sensor that displays this character at
multiple sites separated by a molecular scaffold, selective sensors can be made that
will display fluorescence in the presence of particular carbohydrates or polyols.30
An internal charge transfer based saccharide sensor with an aniline fluorophore was
created by the James group in 2001 that used a boronic acid group to bind diols.31 The
molecule had a reportedly high shift in observed wavelength upon addition of a
saccharide and so more closely resembled an “on-off” receptor than some previous
efforts.
Figure 6 James’s boronic acid based carbohydrate sensors32
12
13
14
NH
(HO)2B
NH
B(OH)2
NH
B(OH)2
11
It was initially thought that the bond formed between boron and nitrogen in all
boronic acid based fluorescence receptors was quite strong, even covalent, but this
was later shown to be incorrect. In some receptors the B-N bond has been shown to
be very weak, much like a typical hydrogen bond.33 This was further explored in a
2004 study by the James group.32 Three boronic acid based saccharide sensors were
made (Figure 6) only one of which had the potential to form a B-N bond of any kind,
and the fluorescence outputs were measured. All three compounds were tested for
fluorescence changes upon binding with D-glucose, D-fructose, D-mannose and D-
galactose. All compounds showed an almost equal increase in fluorescence intensity
upon addition of the sugars with fructose producing the strongest response. The
fluorescence increase in this case is due to the restoration of the aniline fluorophore.
The normally fluorescent aniline had its fluorescence quenched because it was bound
covalently to the phenylboronic acid which acted as an electron acceptor due to
boron’s potentially vacant p orbital. So when the aniline became locally excited it
could transfer charge internally instead of emitting light. When the boronic acid then
changed to the bound anionic form upon binding to a saccharide, it was no longer
electron withdrawing and the fluorescence was restored. This demonstrated another
method for how a fluorescence “on” site can be produced, and also showed that the
spacial proximity of the boron to the nitrogen is not always an important aspect of
fluorescent boronolectin design so long as there is a conjugated system to transfer the
charge (Figure 7).
12
Figure 7 Illustration of the internal charge transfer effect due to boron’s vacant orbital
In 2005 the Houston group reported the successful selective sensing between inositol
epimers by a bis(boronate) fluorescence sensor, originally developed by Shinkai et al.,
this demonstrated the production of a boronic acid based fluorescence sensor with a
potential real-world application.34,20 Serum concentrations of a particular drug could
be determined by using a boronolectin 15 potentially enabling more adept monitoring
of a drug treatment. As can be seen (Figure 8), this boronolectin possessed two
binding sites to enable a higher degree of selectivity.
Figure 8 Bis(boronic acid) fluorescence sensor34,20
Binding enhanced the B-N interactions and restored fluorescence by eliminating the
quenching ability of the nitrogens. Upon binding to inositol an increase in fluorescence
was observed, and because there were two binding sites, two – position binding to a
carbohydrate caused a different spatial arrangement due to the lack of rigidity present
in the molecule. This altered the observed fluorescence when comparing 1:1 and 1:2
15
NH
B(OH)2
Internal Charge Transfer
N
OMe
OMe
N
B(OH)2
B(OH)2
13
receptor/substrate binding. Since only the targeted polyol could bind with a 1:1 ratio,
selectivity was achieved. Armed with the knowledge of how to create fluorescent “on”
and “off” sites, and with previous working examples of fluorescent boronlectins, it can
be possible to design fluorescent receptors for a specific target.
1.4 Specific Target: Sialic Acid
N-Acetylneuraminic acid, also known as Neu5Ac or sialic acid, is a mammalian
carbohydrate that is present as part of many bodily tissues and fluids. It was Alfred
Gottschalk who wrote the first book on the carbohydrate published in 1960 35 and he,
with Gunnar Blix and Ernst Klenk, coined the term “sialic acid”.36 37 Like KDO, a
carbohydrate that will be discussed later, sialic acid possesses a pseudo -
hydroxycarboxylic acid structure at its anomeric center. Sialic acid is present in
sialoglycocongugates on cell surfaces, intracellular membranes, and is an important
component of serum and mucous membranes. Sialic acid performs many functions
but most importantly it is involved in cellular and molecular recognition. It is a
necessary component of the receptors for endogenous hormones and cytokines as
well as pathogenic toxins, bacteria, viruses and protozoa.38
Figure 9 Sialic acid
16
O
OHHO
HO
OHH
H HO
RCO2H
14
It is thought that sialic acid is also involved in tumour metastasis due to its masking
properties to reduce immune response.39 It is the terminal galactose residues that
would normally serve to inhibit cell growth and proliferation that are masked, which
enhances tumour proliferation.40 Metastatic tumour cells are sialylated to a
significantly higher degree than healthy cells. It is thought that the cell surface
terminal sialic acid residues might be detectable through the use of a tuned
boronolectin fluorescence detector. This could provide a tumour detection strategy.
As well as coating the cell surface of metastatic tumour cells, free sialic acid is present
in higher concentrations in the blood serum of cancer patients.41 By measuring levels
of sialic acid in blood, cancerous states can be detected and the time-course of the
disease can be monitored. It has been shown that, in the case of cancer of the uterine
cervix, free sialic acid levels when measured in plasma is a superior biomarker as
compared to serum-bound forms of the carbohydrate.42 Sialic acid levels are also
raised in other cancers ,43 Salla’s disease, 44 cardiovascular disease,45 renal disease,46
liver disease,47 and alcoholism.48 It has been stated that by improving the selectivity of
the methods for detecting free sialic acid in human blood that it could become a
clinically useful biomarker for routine testing.49 50
Current methods for detecting free sialic acid in human plasma include the
resorcinol/orcinol methods and the thiobarbituate method or Warren assay. One of
the first such methods for detecting sialic acid was the resorcinol method.51 A detailed
procedure for measuring sialic acid in human serum using resorcinol was published in
1964,52 but since this method quantifies sialic acid present in precipitated
glycoproteins it lacks specificity for measuring free sialic acid. Another method used
15
for the detection of sialic acid is the orcinol method.53 This method was superseded
because it suffered from intereferences from hexoses, pentoses and uronic acid. The
resorcinol method for detecting sialic acids remained the most effective until the
thiobarbituate method was applied. The thiobarbituate method or Warren assay was
described first for use on 2-deoxyribose which was oxidised to produce malonaldehyde
which in turn reacted with the thiobarbituate.54 A modified version of this method
was later applied to 2-deoxy-keto sugar acids.55 This method was further modified and
applied to sialic acid to produce a 12 fold improvement over the resorcinol method.56
This method was also so selective for free sialic acid over bound sialic acid that to
measure total sialic acid present in biological samples, the samples had to first be
heated to 800 C for 1 hour in sulphuric acid to release the bound sialic acid. This
represents the first method for detecting free sialic acid selectively, and it was far from
perfect. Its main downfall was that it was a labour intensive process and although
sensitive, its sensitivity could be further improved. This is an area where fluorescent
boronolectins would make ideal agents for the specific sensing of unbound sialic acid.
1.5 Specific Target: KDO
Keto-3-deoxy-2-octulosonic acid (KDO, 17) is a carbohydrate residue that is present as
part of the lipopolysaccharide in Gram-negative bacteria.57 This includes notably
Helicobacter pylori, Escherichia coli and Haemophilus influenzae as well as many other
pathogenic strains of bacteria.58,59 In all cases, KDO is present in the core region of the
outer membrane of Gram-negative bacteria. KDO is essential since it forms the link
between lipid A and the LPS structure that makes up the outer membrane. Different
bacteria incorporate KDO into their LPS core in different ways. For example, E.coli
16
requires only KDO2-lipidA synthesized via a bifunctional KDO transferase as the
minimum essential LPS structure under laboratory conditions, whereas H. influenzae
requires KDO-phosphate-lipid A synthesized using a monofunctional KDO transferase
and a KDO kinase.60 It has been hypothesized that modified forms of KDO may provide
targeted antibiotic solutions in the form of new generation carbohydrate based
antibiotics.61
Figure 10 KDO
A recent review covers this topic in detail and concludes that KDO is critical for
bacterial viability.62 Selective binding of KDO has also been implied as a mechanism for
antibacterial activity related to Gram-negative bacteria.63,64 If binding of KDO is
possible, and if KDO is present in the LPS in a form that can be bound using boronic
acid based synthetic lectins, it is possible that a detection technique could be
developed for fluorescently detecting Gram-negative bacteria. A boronic acid based
synthetic lectin that shows selectivity for KDO could potentially be a viable
antibacterial selective for gram-negative bacteria. Since KDO is not present in
mammalian cells and has features that are unique compared to other biologically
occurring carbohydrates, this makes it a suitable target for selective binding from a
receptor design viewpoint.
17
O
OHHO
HO
HO
OH
COOH
17
1.6 “Designer” Boronolectins
Boronic acid sensing of the monosaccharide sialic acid (Neu5Ac) is something that has
been attempted by several groups and in a 1998 example this was achieved using 18
attached to a polymeric scaffold.
18
Figure 11 Taylor and Smith’s boronic acid based fluorescence sensor moiety used in a
polymeric sensor65
The selectivity of this fluorescence sensor for sialic acid demonstrated by Taylor and
Smith provides an example of a library based screening approach to the generation of
a selective boronic acid based sensor.65 The goal was to create a fluorescence sensor
that could detect sialic acid in physiological concentrations of glucose. They were
successful in achieving this but the sensor was also responsive for fructose unless the
poly(allylamine) polymeric scaffold was loaded with a certain amount of the
compound before being further loaded with certain amounts of two other components
(4-imidazolacetic acid and 4-hydrobenzoic acid). This caused a slightly higher affinity
for sialic acid than for fructose due to a speculated induced fit of the polymer to the
N
O
H
B(OH)2
18
sialic acid. It was thought that after the chelation of a sialic acid side chain to the
boronic acid, secondary interactions occurred with other functional groups attached to
the polymer chain. This is an example of tuning a particular compound to have a
selective response for a target carbohydrate and demonstrated how it is possible to
generate a custom made sensor. A non-polymeric example of this concept appeared
in 2004 where the tuning of a boronic acid based sensor was successful in creating the
first true boronolectin to target a cell-surface oligosaccharide.
Wang developed a selective fluorescence sensor incorporating two boronic acid sites.
The carbohydrate sensor 19 was made with R being one of many possible linkers to
provide various distances between the two binding areas. This compound was the first
“designer” boronolectin shown to be functional on a cell surface. By using different
linkers the selectivity of the sensor could be tuned to detect sialyl LewisX. This
boronolectin was then demonstrated to have significance when applied to HEPG2,
HEP3B and COS7 cells and fluorescence was visible unless neuraminidase or fucosidase
treatment was applied implying that sialyl LewisX was the target. This is the first
documented example of cell surface carbohydrates being detected by a small organic
fluorescence sensor. Previously an example of a boron based sensor with a targeted
application appears in a 2001 article by Sugasaki et al.66 A double decker and a meso-
meso linked porphyrin was created that incorporated a boronic diol to bind Lewis
oligosaccharides. This was an example of a compound that had potential applications
in the detection of tumours that overexpress sialyl LewisX although did not display this
kind of selectivity to a high degree.
19
Figure 12 Wang’s “designer” boronolectin
Sialic acid (16) was targeted in 2005 describing the way in which a phenylboronic acid
binds to the carbohydrate.67 It was found that sialic acid interacts with phenylboronic
acid and can interact through the -hydroxycarboxylic acid (C1,C2) or the glycerol
moiety (C7-C9). It also was discovered that C7,C8 binding is unlikely due to the
unfavourable erythro configuration. The pH dependency of these binding positions
was also discussed and it was stated that the -hydroxycarboxylic acid of sialic acid will
preferentially bind at pH 2-8 whereas the other binding sites are favoured at pH 8 or
higher. Although it was stated the C7,C8 binding is unfavourable, previous work
demonstrates that due to B-N interactions the C7,C8 complex may be the major
complex in some examples.68 In 2003, 3-(propionamido)phenylboronic acid was
chosen as a model compound for complexation to sialic acid and a conformational
diagram showed that the C7,C8 bound complex was the major product in aqueous
media. It was suggested that this is due to interactions between boron and the amide
of sialic acid that facilitate the formation of the sp3 hybridized boron complex.68
19
N
(HO)2B
NH
H2C R
H2C N
H
N
B(OH)2
20
OOR
O
X
HN
O
OO
HO
OOR
O
X
HN
O
OH
O
OB
Ar
OOR
O
X
HN
O
OOO
OOR
O
X
HN
O
O
HO
O
B
Ar
B
Ar
B
Ar
In 2006 the Duggan group reported on the binding of sialic acid as well as sialic acid
derivatives by an aryl boronic acid.69 In their work the bioavailability of carbohydrate
derived drugs was shown to be improved by a lipophillic boronic acid that was used as
a molecular chaperone. In the paper they discuss possible binding modes of sialic acid
derivatives to an aryl boronic acid. The sialic acid derivatives used included Relenza®
with variations at C1 and C4 (Figure 13).
7,8-ester
8,9-ester
7,9-ester
7,8,9-ester
Figure 13 Possible binding modes of a boronic acid to a sialic acid derivative (R = H or Me, X
= OH, N3 or NHC(=NH)NH2)69
21
A recent example where a boronic acid based sensor was used to give a measurable
output when sialic acid was applied was provided by the Hindsgaul group in 2007.70 In
this approach, rather than designing a fluorescent boronolectin, an indicator dye was
bound to the previously reported71 o-hydroxymethylphenyl boronic acid. Through
their process, terminal glycosylation of a glycoprotein bound to resin beads could be
observed by the naked eye. Starting with hydroxylamine functionalized resin beads it
was shown that the addition of galactose, released by -galactosidase from a
glycoprotein, could produce the aldoxime with galactose now bound to the resin in
open chain form. To this was added the strongly coloured boronic acid dubbed TMR-B
made in a single step from tetramethylrhodamine (TMR) and o-hydroxymethylphenyl
boronic acid. The TMR-B was shown to bind to the bound glycerol and after washing
the white resin beads had turned a red colour, indicating the presence of galactose
(Figure 14). Other carbohydrates were combined in the system and activity was shown
also for fructose, sialic acid and GlcNAc. By cleaving TMR-B after a washing step, the
affinity of the boronic acid for the different carbohydrates could be measured by
comparing relative fluorescence (Figure 15).
22
Figure 14 TMR-B bound to the glycerol tail of
the galactose aldoxime formed on resin bead 70
Figure 15 Relative response of the different
sugars tested 70
O
AcHNO
N
OH
OH
OH
O B
O
O
NH
TMR
23
1.7 Catalysis
An additional aspect of this thesis describes the manipulation of boron-hydroxycarboxylic
acid complexes to catalyse functional group transformations. Boronic acids catalyse a range
of organic reactions, including Mukaiyama aldol reactions,72 Oppenauer oxidations,73
amidation of carboxylic acids,74 imine hydrolysis,75 cyclisations,76 arylations of phenols,77
cyclodehydration78 and the Hantzsch reaction.79 In some reactions the boronic acid group is
used as a non-activating protecting group, in others it is an activating group that directs
reactions that otherwise could not take place.30 Boric acid itself can be used to catalyse
some esterification reactions and the mechanism of these have been studied in the past.80
Esterification reactions make up an important part of organic chemistry and are important
tools in many syntheses. Boronic acids produce milder reaction conditions than other acid
catalysts used in esterification. They can also provide selective esterification meaning that
when an acid like malonic acid is used the ester produced will be predominately in its
monoester form rather than the diester that might be expected. It was first described in a
2004 paper by the Houston group that the boric acid catalyst itself forms esters with alcohol
groups available in the reaction mixture, this new compound then becomes the catalyst.81
H. Yamamoto then went on to propose why a faster reaction can sometimes occur when the
reacting alcohol itself is not used as the solvent for the reaction.82
24
Figure 16 Mechanistic rationale of boric acid catalysed esterification of an -hydroxycarboxylic
acid81
1.8 Project Aims
The purpose of the work included in this thesis is to:
Generate boronolectins for binding to a specific carbohydrate substrate
Generate carbohydrate and carbohydrate-like molecules for use in binding assays
Further explore boric acid and boronic acids as reaction catalysts
The main focus of the work included in this thesis is to synthetically produce a boronic acid
containing compound for use as a molecular fluorescence sensor selective for
monosaccharides. This work can roughly be divided into 1) The generation of fluorescent
boronolectins, and 2) The generation of ligands for binding to a molecular sensor. A
reaction catalysed by boric acid is further explored in another aspect of the project involving
ROH
OH
O
RO
O
O
B
OR'
RO
O
O
B
R'O
ROH
OH
O
OR'
B(OR')3
R O
O
B
O
OH
H
X
25
the generation of salicylate esters, with potential antibacterial activity. Amide bond
formation reactions also produced interesting results that were further explored,
uncovering a novel use for boronic acids.
26
Chapter 2
Synthesis of Carbohydrate
Ligands
27
2.1 Overview
This chapter is concerned with the synthesis of ligands to be used in carbohydrate sensing
assays. In order to determine the positions on the carbohydrates which are involved in
binding to the boronic acid based receptors, modified forms of some carbohydrates were
made for assays. There are numerous ways in which these monosaccharides can be
modified. One method of producing a modified form of a carbohydrate, in the case of
carbohydrates that possess an -hydroxycarboxylic acid, is by esterification. A series of
esterifications are reported and the structure and binding functions of the targeted
carbohydrates are discussed. The synthesis and purification of the eight carbon sugar-acid,
KDO is reported. Another series of esterification reactions utilised to make salicylate esters
is reported, these compounds were made with the purpose of having potential antibacterial
or antifungal activity.
2.2 Boronolectins, KDO and Sialic Acid
In biological systems the protein class known as lectins play an important role. Biological
lectins are carbohydrate binding proteins and their purpose is to bind to specific
carbohydrate structures with a high degree of selectivity. Among many other roles they are
involved in the binding of glycoproteins to cell surfaces, they are responsible in part for
controlling protein levels in blood, and take part in key roles that apply to the function of
the human immune system. The term “boronolectins”83 refers to synthetic lectins which
display one or more boronic acids as binding points for diols, polyols and -
hydroxycarboxylic acid-like structures including the types found on carbohydrates.84 This
field of research is rapidly growing as the carbohydrate binding ability of boronic acids is
28
explored and enhanced through the inclusion of multiple binding sites as well as changes in
their structural geometry for improved selectivity. There is a need for more specific boronic
acid based receptors to be designed and explored primarily due to their potential uses,
ranging from enzyme inhibition, to antibacterial agents, to the fluorescence aided sensing of
clinically relevant carbohydrate levels. These boronolectins could even have the potential
to be used in the sensing of surface bound carbohydrates in order to evaluate diseased
states.
In order to observe to affinity of a boronolectin for a particular carbohydrate it is necessary
to perform assays, these could be fluorescence assays, to quantify its affinity. By modifying
the targeted carbohydrate at certain positions it is possible to remove potential binding
sites. In this way, by recording changes in fluorescence output, binding positions can be
identified. One method for modifying carbohydrates that possess an -hydroxycarboxylic
acid is to esterify at that position, thus removing the -hydroxycarboxylic acid as a potential
binding position. In 2004 the Houston group reported the chemoselective esterification of
-hydroxycarboxylic acids in alcohol solvents using boric acid as a catalyst (Figure 17).81
Figure 17 Boric acid catalysed esterification of an -hydroxycarboxylic acid.
R
HO
O
OHR'OH
B(OH)3
10-20 mol%
R
HO
O
OR'
29
It was reported that -hydroxycarboxylic acids could be esterified by using boric acid as a
catalyst without significant esterification of other carboxylic acids which do not possess the
-hydroxyl group. The procedure used involved stirring in the alcohol solvent overnight at
room temperature, a relatively mild procedure well suited to carbohydrate chemistry. Here
we have applied this chemistry to the chemoselective esterification of sugar acids. This is
important in that it provides information about the affinity of boron for particular binding
sites. One important example of an acidic sugar is KDO, which is found in Gram-negative
bacteria as part of the lipopolysaccharide.57
The incorporation of KDO into the lipopolysaccharide has been shown to be a vital step in its
synthesis by the bacteria and vital to its growth.85,86 By preventing the incorporation of KDO
into the lipopolysaccharide it is hypothesised that an antibacterial effect may be produced.
The selective binding of KDO has already been implied as a possible mechanism for
designing new antibiotics.63,64 Since KDO does not occur in eukaryotic cells, targeting the
carbohydrate selectively could potentially lead to physiologically active antibiotics. In
targeting KDO for binding with a boronic acid based agent it is important to consider its
molecular structure. KDO is an eight carbon monosaccharide and possesses at least 3
potential diol or -hydroxycarboxylic acid binding sites (Figure 18).
30
Figure 18 Potential boronic acid binding sites present on KDO
By structurally modifying KDO potential binding sites could be removed, in this way the
binding character of a receptor could be observed. By methylating the carboxylic acid to
form an ester or by removing or modifying one of the hydroxyl groups this could be
achieved. Shown in Figure 19 is the mechanism for the synthesis of KDO starting from
oxalacetic acid and D-arabinose. The reaction involves a base-catalysed aldol condensation
between the two reagents, followed by an in situ decarboxylation upon acidification of the
reaction mixture. In this way KDO can be obtained in around 30% yield.87 KDO was not the
only monosaccharide that is discussed in later chapters, another targeted carbohydrate with
biological importance was sialic acid.
O
OHHO
HO
HO
OH
C
O
OH
B
B
B
31
Figure 19 Mechanism for the Cornforth method applied to KDO synthesis
Sialic acid (16) is a nine carbon monosaccharide possessing an N-acetyl group, also known as
N-acetylneuraminic acid. In modern commonly used nomenclature the term “sialic acid”
refers to this carbohydrate, although in older writings dated around mid-last century “sialic
acid” can refer to a number of variations of this sugar. The purpose of targeting sialic acid
was to detect the carbohydrate in solution with a high degree of selectivity, and to produce
a measurable change in fluorescence output upon binding to a boronic acid based receptor.
Sialic acid, like KDO, has multiple ways in which it might bind to a boronolectin.
16
The glycerol tail comprised of C7, C8 and C9 of sialic acid could potentially bind to a boronic
acid in 4 possible modes. The C8, C9 and the C7, C8 hydroxyl groups could bind as 1,2 diols.
O
OHHO
HO
OHH
H HO
RC
R = NHAc
O
OH
O
HO
OH
OH
HO
O
O
O
O
OOH
OHHO
HOOH
O
O
HO
O
O
OH
OHHO
HOOH
O
O
HO
H
OH
OHHO
HOOH
OH
O
HO
O
OHHO
HO
HO
OH
C
O
OH
H
32
Also the C7, C9 hydroxyl groups could bind as a 1,3 diol unit, or all three of the glycerol
hydroxyl groups could bind to the boronic acid producing a charged boronate species. At
the other end of the molecule there is the pseudo -hydroxycarboxylic acid moiety that
could bind to a boronic acid favourably at a lower pH, also the hydroxyl group at C4 could
potentially be involved in binding as well. By modifying the carbohydrate at any of these
positions we can eliminate the potential for binding at these positions, thus exploring the
binding mode to the receptor.
2.3 Project Objectives
Carbohydrate targets had to be prepared for this study so that the binding affinity of the
boronic acid based receptors could be observed. The objectives of this chapter were to: (1)
Produce crystalline KDO of sufficient purity to be used in binding studies, (2) Produce
modified carbohydrate derivatives of both KDO and sialic acid to further explore binding
behaviour, (3) Examine boric acid as a catalyst for the esterification of -hydroxycarboxylic
acids and sialic acid.
2.4 Preparation of Carbohydrate Derivatives
For the purpose of generating a library of compounds for use in fluorescence experiments
some reactions were performed using boric acid as a catalyst for the esterification of sugar
acids. We also explored this chemistry in other systems. The starting compounds that were
used in the attempted esterifications were sialic acid, KDO, glucuronic acid, quinic acid and
KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid). These experiments, summarised
33
later in Table 1, demonstrate boric acid’s ability esterify carboxylic acid groups present on
carbohydrates and also shows cases where it is not an ideal catalyst. By generating
modified forms of carbohydrates this creates substrates for use in further fluorometry. The
results of assays that were run using modified carbohydrates gave information relevant to
the binding positions being used by the receptors tested. If a modified carbohydrate gave
an altered response compared to the parent compound due to the removal of a possible
binding site, it could be surmised that the site was involved in the binding event.
2.4.1 KDO Synthesis
KDO (17) was synthesized for this project from D-arabinose (20) and oxaloacetic acid (21)
using the method reported by McNicholas et al.,88 which was an implementation of the
Cornforth method.89 Crystallization of KDO was achieved from a viscous aqueous solution of
KDO with trace amounts of mixed organic solvents of differing polarities. The mixture
initially was dilute and included mostly water with ethanol. To this was added ethyl acetate
and hexanes before filtering and concentrating to viscous syrup by vacuum at room
temperature before cooling to produce crystals that could be washed with ethanol to give
pure KDO. The ability to produce KDO of high purity allowed many experiments to be
conducted and ensured the validity of our research. Modified forms of KDO can be
produced by using starting materials other than arabinose.87
34
20 21 17
Scheme 1
KDO (17) has multiple sites for potential boronic acid binding, one of these sites is the -
hydroxycarboxylic acid. An attempted reaction was performed to esterify KDO to make KDO
methyl ester 22 as per Houston et al., 2004 (Scheme 2). This reaction did not proceed to
produce KDO methyl ester, starting material remained unchanged even after 3 days with
heating at 500C. It is unknown why this reaction did not proceed despite KDO possessing
the necessary -hydroxycarboxylic acid, although it is possible that with an abundance of
potential boron binding sites boron has bound preferentially at another position on the
carbohydrate rendering it ineffective as a catalyst.
17
Scheme 2
22
Another attempted reaction was performed to esterify KDO (17) to 2-phenylethanol (23) in
order to make KDO phenylethyl ester (Scheme 3). This reaction did not proceed to produce
KDO phenethyl ester, starting material remained unchanged after 5 days stirring at room
temperature. Again, it is unknown why this reaction did not proceed despite KDO
possessing the necessary -hydroxycarboxylic acid.
O
OH
HO
HO
OH
+
O
O
HO
O
OH
1.NaOH
2.H+
H2O
OCOOH
OH
HO
HO
OHHO
CO2
O
OHHO
HO
HO
OH
COOH
MeOH
B(OH)3
O
OHHO
OH
HO
OH
COOMeX
35
Scheme 3
KDN (25) was another carbohydrate that was evaluated as a potential substrate in this work.
KDN is structurally similar to sialic acid, with the C-5 N-acteyl group in sialic acid replaced by
a hydroxyl group in KDN, adding to the potential complexity associated with interactions
with boronic acids. An esterification reaction was performed with methanol and boric acid,
this reaction proceeded to produce the product 26 in high yield (>90 %) (Scheme 4). Only
trace amounts of starting material were visible by 1H NMR and so the crude product was
used for assays after the boric acid was removed by vacuum as trimethyl borate (b.p. 68-
690C). Trace starting material was deemed to be acceptable due to the qualitative nature of
the carbohydrate assays, for comparison purposes.
KDN: R = OH (25) Sialic acid: R = NHAc (16)
26: R = OH 27: R = NHAc
Scheme 4
Sialic acid (16) is a carbohydrate of particular interest in this thesis. Modified versions of
sialic acid were used to examine its binding mode. An esterification reaction was performed
17
23
24
O
OHHO
HO
OHH
H OH
RCO2H
MeOH
B(OH)3
O
OHHO
HO
OHH
H OH
R
O
OMe
OHO
OHHO
HO
HO
OH
COOH
CH3CN
B(OH)3
X+
O
OHHO
HO
HO
OH
O
O
36
with methanol using boric acid as a catalyst. This reaction proceeded to give the product 27
in high yield (>90 %) (Scheme 4). Only trace amounts of starting material were visible by 1H
NMR and so the crude product was used for assays after the boric acid was removed by
vacuum as trimethyl borate (b.p. 68-690C). Heating the reaction at 50°C produced a better
yield in a shorter time span.
A reaction was performed as an attempted esterification using glucuronic acid (28) and
methanol with boric acid as a catalyst (Scheme 5). This reaction did not proceed to produce
the desired product. Starting material remained unchanged after 24 hours stirring at room
temperature. This reaction did not proceed to give the ester 29, most likely due to
glucuronic acid not possessing an -hydroxycarboxylic acid. This demonstrates that the -
hydroxycarboxylic acid must be less reactive to boron catalysis.
Scheme 5
For completeness glucuronic acid (28) was revisited as a starting material for an
esterification reaction, this time using benzyl alcohol as the co-reagent and boric acid (10
mol %) as the reaction catalyst (Scheme 6). Again, this reaction failed to produce the ester
product 30.
28
29
OCOOH
HOHO
OH
OH
MeOH
B(OH)3
XO
COOMe
HOHO
OH
OH
37
Scheme 6
Quinic acid (31) was another carbohydrate that was assayed for binding to boronates in a
later part of the project. Abundant in nature,90 it displays a number of diols available for
potential boronic acid binding. By modifying one of the alcohol groups the potential for
binding to a boronic acid at that position is eliminated. An esterification reaction was
performed using quinic acid and methanol with boric acid as a catalyst. This reaction
proceeded to produce the product 32 in excellent yield (>90 %) (Scheme 7). Only trace
amounts of starting material were visible by 1H NMR and so the crude product was used for
assays after the catalytic boric acid was removed by vacuum as trimethyl borate (b.p. 68-
690C).
Scheme 7
Another attempted reaction was performed as an attempted esterification using quinic acid
(31) and 2-phenylethanol (33) with boric acid as a catalyst in order to make quinic acid
phenylethyl ester 34 (Scheme 8). This reaction did not proceed to produce quinic acid
phenethyl ester, starting material remained unchanged after 24 hours stirring at room
28 35 30
31 32
HO
HO
OH
OH
O
OHMeOH
B(OH)3
HO
HO
OH
OH
O
O
OCOOH
HOHO
OH
OH
B(OH)3
XO
COOBn
HOHO
OH
OH+
CH3CN
OH
38
temperature. This reaction did not proceed despite quinic acid possessing the necessary -
hydroxycarboxylic acid. It is possible that the cis-vicinal diol, an additional binding site for
the boron, prevented the reaction from proceeding.
Scheme 8
It was found that changing the reactant alcohol from 2-phenylethanol (33) to benzyl alcohol
(35) also failed to furnish the desired quinic acid benzyl ester 36 (Scheme 9). This could
again be attributed to the cis-vicinal diol interfering with the reaction.
Scheme 9
In the case of the esterifications performed using larger alcohols, a different procedure was
used. Rather than using the high molecular weight, high boiling point alcohols as solvents
for the reactions, the alcohols were used in a 1:1 stoichiometric ratio with the carbohydrate
to be esterified. In these cases acetonitrile was used as the solvent as this potentially will
31
33
34
31
35
36
OH
CH3CN
B(OH)3
X+
HO
HO
OH
OH
O
OH HO
HO
OH
OH
O
O
OH
CH3CN
B(OH)3
X+
HO
HO
OH
OH
O
OH HO
HO
OH
OH
O
O
39
speed the reaction rate. Shown in Figure 20 is the mechanism first proposed by Yamamoto
and co-workers in 2005 to explain the rate increase seen when the molar amount of
reactant alcohol was decreased in a mixture containing an aprotic co-solvent.
Figure 20 Slow/Fast mechanism relevant when using equimolar amounts of reactants91
The reactions performed with larger alcohols were shown to have a better chance of
reacting. Although in our case there was no product formed with the heavier alcohols, this
was most likely due to the extra steric bulk of benzyl alcohol and 2-phenylethanol as
compared to the low steric bulk of methanol.
O
B
OO
R1
OR2
OR2
OH
R1 CO2H
2 R2OHO
B
OO
R1O
O
O
R
R1 CO2R2
OH
very fastvery slow
40
Table 1 Acidic carbohydrate esterification using boric acid
Acid Alcohol Product Yield
KDO
Methanol
CH3OH
NR
0%
KDO
2-Phenylethanol
NR
0%
KDN
Methanol
CH3OH
26
>90%
Neu5Ac
Methanol
CH3OH
27
>90%
Glucuronic Acid
Methanol
CH3OH
NR
0%
Glucuronic Acid
Benzyl Alcohol
NR
0%
O
OHHO
HO
HO
OH
COOH
O
OHHO
HO
HO
OH
COOH
OH
O
OHHO
HO
OHH
H OH
R
O
OH
R = OH
O
OHHO
HO
OHH
H OH
R
O
OMe
R = OH
O
OHHO
HO
OHH
H OH
R
O
OH
R = NHAc
O
OHHO
HO
OHH
H OH
R
O
OMe
R = NHAc
OCOOH
HOHO
OH
OH
OCOOH
HOHO
OH
OHOH
41
Quinic Acid
Methanol
CH3OH
32
>90%
Quinic Acid
2-Phenylethanol
NR
Under
5%
Quinic Acid
Benzyl Alcohol
NR
0%
Shown in Figure 21 is the mechanism for the esterification of the -hydroxycarboxylic acid,
sialic acid. This carbohydrate was esterified with a greater than 90 % conversion rate using
~50 mol % boric acid as a catalyst in methanol solvent with mild heating overnight. KDN
reacted with similar conversion with only 25 mol % boric acid catalyst under the same
conditions at room temperature. Quinic acid performed just as well with only 10 mol %
boric acid, also at room temperature. The mixed anhydride species shown on the left of the
diagram can form in the absence of protic solvents, but is unlikely to form in the presence of
methanol.81
HO
HO
OH
OH
O
OH
HO
HO
OH
OH
O
OHOH
HO
HO
OH
OH
O
OH
OH
HO
HO
OH
OH
O
O
42
O
OHHO
HO
OHH
H HO
RC
R = NHAc
O
OH
B(OMe)3
O
OHHO
HO
OHH
H HO
RC
O
OMe
O
OHO
HO
OHH
H HO
RC
O
O
B OMe
MeO
O
OHHO
HO
OHH
H HO
RC
O
OB
O
OH
HX
Figure 21 Proposed mechanism for boric acid catalysed esterification of sialic acid
Following on from the work concerning esterification reactions involving quinic acid, further
exploratory esterifications were attempted using galactose (37) and galactose-like
carbohydrates. The purpose of this work was to further examine boric acid as a versatile
catalyst. Following the attempted boric acid catalysed esterification reaction with simple
alcohols, it was decided to explore the potential and versatility of this approach. Using
galactose as a template we reasoned that boric acid, through an ability to form
exchangeable bonds with alcohols, could form a cyclic ester with the galactose cis-3,4-diol.
This complex (Figure 22) would then bring the -hydroxycarboxylic acid component into
close proximity to the C-6 alcohol in galactose, and could therefore facilitate a
regiochemically specific reaction at the C-6 hydroxyl. In this way it was thought that
galactose could have a favourable configuration to enable an esterification to take place.
43
Figure 22 Proposed complex formed between boric acid, mandelic acid and galactose
No reaction was recorded for the attempted reaction between mandelic acid (38) and
galactose (37, Scheme 10). After this we moved on to attempt the reaction with 2-deoxy-D-
galactose.
38 37
Scheme 10
A reaction was attempted using 2-deoxy-D-galactose (39) and mandelic acid (38, Scheme
11). This reaction did not proceed at high yield with stirring for 24 hours at room
temperature. A crude 1H NMR suggested a slight conversion to the product, but more
vigorous reaction conditions would likely be required to produce a high yield, boric acid may
not be an ideal catalyst for this reaction.
Scheme 11
38
39
O
OH
O
OHO
OH
BO
O
O
HO
O
OH
+
B(OH)3
CH3CN
NRXO
OH
HO
OH OH
OH
HO
O
OH
OHO
OH OH
OH+
B(OH)3
CH3CN
NRX
44
Table 2 Attempted mandelic acid – neutral carbohydrate esterification using boric acid
Acid Alcohol Product
Mandelic acid
Galactose
No
Mandelic acid
2-deoxy-galactose
No
The two reactions shown in Table 2 were performed using boric acid as a catalyst and did
not yield product. NMR spectroscopy of the reaction crude in these cases suggests a small
amount product formation, but purification was not able to be conducted due to the small
amount of potential product. Boric acid was not an effective catalyst for these reactions.
KDO would not form an ester with methanol or 2-phenylethanol using boric acid as a
catalyst, despite having the necessary -hydroxycarboxylic acid. The lack of reactivity could
be due to its instability under acidic conditions. Glucuronic acid as expected was not
successfully esterified due to the lack of an -hydroxycarboxylic acid. Although quinic acid
readily formed the methyl ester, a benzyl ester could not be formed using this method.
Likewise 2-phenylethanol could not be esterified to quinic acid in acceptable yield.
HO
O
OHO
OH
HO
OH OH
OH
HO
O
OHO
HO
OH OH
OH
45
Following on from the esterification of carbohydrates was the esterification of salicylic acid.
Although there is little recent work published on the topic, salicylate esters have been found
to control populations of bacterial, worm and fungal organisms.92 Salicylic acid is also
considered a plant hormone that can provide protection to threats and pathogens, and the
volatile ester, methyl salicylate is used by plants as a signaling hormone to nearby
neighbours warning of a potential threat.93 A review was published in 2000 covering the
topic of the effects of salicylate on bacteria.94 In the review it is stated that the presence of
some salicylates in the human body can produce effects including antibiotic resistance,
antibiotic susceptibility and can affect bacterial virulence factors. Therefore a series of
reactions were performed to produce salicylate esters with potential antibacterial
properties.
Salicylic acid (42) and 2-phenylethanol (33) were combined in acetontrile with 10 mol % of
boric acid and heated to reflux for 16 hours to produce the ester product 40 in a 17.3 %
yield (Scheme 12). After flash column chromatography was performed on silica using neat
ethyl acetate as the mobile phase, the product could be brought to high purity by
crystallization.
46
B(OH)3
OH
O
O
HO
OH
B(OH)3
OH
O
O
B(OH)3
OH
O
O
OH
n = 8
n = 8
B(OH)3
OH
O
O
OH
OH
O
OH
6.3%
55%
17%
35%
40
41
33
42
43
44
45
46
47
Scheme 12 The esterification of salicylic acid using 10 mol% boric acid as a catalyst in refluxing
acetonitrile solvent
The same procedure was used to produce ester product 41 in a 6.3 % yield. Likewise 44 and
47 were produced from decyl alcohol (45) and propargyl alcohol (46) respectively with yields
of 34.6 % and 55.0 %. Due to the highly crystalline nature of compound 47 crystals were
able to be grown from toluene at a reduced temperature ~40C of high quality for
crystallography studies. This was published in Acta Crystallographica Section E on 16
December 2009.
49
asymmetric unit. The transoid propynyl ester groups are coplanar with the 2-hydroxy-
benzoate group with maximum deviations of -0.3507 (3) and 0.1591 (3) Å for the
terminal carbons, with intramolecular O—H∙∙∙O hydrogen bonding providing rigidity to
the structure and ensuring that the reactivity of the alkyne is not compromised by
steric factors. The propynyl group forms intermolecular C—H∙∙∙O interactions with the
phenolic O atom. Supramolecular chains along the b axis are found for both molecules
with links by weak O—H∙∙∙O intermolecular interactions in the first independent molecule
and C—H∙∙∙O interactions in the second.
Related literature
For background to Cu(I)-mediated azide-alkyne cyclo-additions, see: Houston et al.
(2008); Wilkinson et al. (2009). For the biological use of salicylates, see: Sox & Olson (1989).
For background to boric acid-mediated esterification, see: Houston et al. (2004, 2007);
Levonis et al. (2007). For stereo-chemistry, see: Wilkinson et al. (2006); Wiberg & Laidig
(1987). For previous synthesis of the title compound and its anti-tumour activity, see: Jung
et al. (1997).
Experimental
OH
O
O
50
Crystal data: C10H8O3; Mr = 176.16; Monoclinic, P21 /c; a = 18.7150 (14) Å; b = 12.7972 (10) Å; c
= 7.2310 (7) Å; = 90.191 (8)°; V = 1731.8(3) Å3; Z = 8; Mo K radiation = 0.10 mm-1; T =
296K; 0.36 x 0.30 x 0.12 mm.
Data collection
Oxford-Diffraction GEMINI S Ultra diffractometer; Absorption correction: multi-scan
(CrysAlis RED; Oxford Diffraction, 2009); Tmin = 0.965, Tmax = 0.988; 10756 measured
reflections; 3081 independent reflections; 1941 reflections with I > 2 (I); Rint = 0.031.
Refinement
R[F 2 > 2 (F 2)] = 0.039; wR(F 2) = 0.098; S = 0.93; 3081 reflections; 235 parameters; H-atom
parameters constrained; pmax = 0.14 e Å-3; pmin = -0.14 e Å-3.
Table 1 Hydrogen-bond geometry (Å, ˚).
D—H∙∙∙A D—H H∙∙∙A D∙∙∙A D—H∙∙∙A
O1—H1∙∙∙O7 0.90 1.86 2.6193 (16) 141
O1—H1∙∙∙O7i 0.90 2.55 3.2081 (17) 130
O11—H11∙∙∙O17 0.90 1.82 2.6007 (18) 144
C10—H10∙∙∙O11ii 0.95 2.38 3.310 (2) 165
C16—H16∙∙∙O17iii 0.96 2.48 3.291 (2) 143
C20—H20∙∙∙O1iv 0.95 2.46 3.340 (2) 154
Symmetry codes: (i) –x + 1; -y; -z; (ii) –x + 2; -y; -z; (iii) –x + 2; y + (1/2); -z + (1/2); (iv) –x + 1; -y; -z + 1.
Data collection: CrysAlis CCD (Oxford Diffraction, 2009); cell refinement: CrysAlis RED
(Oxford Diffraction, 2009); data reduction: CrysAlis RED; program(s) used to solve structure:
SIR97 (Altomare et al., 1999); program(s) used to refine structure:
SHELXL97 (Sheldrick, 2008); molecular graphics: ORTEP-3 for Windows (Farrugia, 1997);
51
software used to prepare material for publication: PLATON (Spek, 2009) and publCIF
(Westrip, 2010).
We acknowledge support of this work by Griffith University, the Queensland University of
Technology, the Eskitis Institute for Cell and Molecular Therapies, and the Institute for
Glycomics.
Supplementary data and figures for this paper are available from the IUCr electronic
archives (Reference: TK2601).
References
Altomare, A., Burla, M. C., Camalli, M., Cascarano, G. L., Giacovazzo, C., Guagliardi, A.,
Moliterni, A. G. G., Polidori, G. & Spagna, R. (1999). J. Appl. Cryst. 32, 115-119.
Farrugia, L. J. (1997). J. Appl. Cryst. 30, 565.
Houston, T. A., Levonis, S. M. & Kiefel, M. J. (2007). Aust. J. Chem. 60, 811-815.
Houston, T. A., Quader, S., Boyd, S. E., Jenkins, I. D. & Healy, P. C. (2008). Acta Cryst. E64,
o1738.
Houston, T. A., Wilkinson, B. L. & Blanchfield, J. T. (2004). Org. Lett. 6, 678-681.
Jung, M., Kerr, D. E. & Senter, P. D. (1997). Archiv. Der Pharm. 330, 173-176.
Levonis, S. M., Bornaghi, L. F. & Houston, T. A. (2007). Aust. J. Chem. 60, 821-823.
52
Oxford Diffraction (2009). CrysAlis CCD and CrysAlis RED. Oxford Diffraction Ltd,
Yarnton, England.
Sheldrick, G. M. (2008). Acta Cryst. A64, 112-122.
Sox, T. E. & Olson, C. A. (1989). Antimicrob. Agents Chemother. 33, 2075- 2082.
Spek, A. L. (2009). Acta Cryst. D65, 148-155.
Westrip, S. P. (2010). publCIF. In preparation.
Wiberg, K. B. & Laidig, K. E. (1987). J. Am. Chem. Soc. 109, 5935-5943.
Wilkinson, B. L., Bornaghi, L. F., Houston, T. A. & Poulsen, S.-A. (2009). Click Chemistry in
Carbohydrate Based Drug Development and Glycobiology: An Update in Glycobiology
Research Trends, edited by G. Powell & O. McCabe, pp. 127-172. New York: Nova Science
Publishers.
Wilkinson, B. L., Bornaghi, L. F., Houston, T. A., Poulsen, S.-A. & White, A. R. (2006). Acta
Cryst. E62, o5065-o5067.
Supplementary materials
Acta Cryst. (2010). E66, o226-o227 (doi:10.1107/S160053680905421X)
2-Propynyl 2-hydroxybenzoate
S. M. Levonis, M. J. Kiefel, T. A. Houston and P. C. Healy
53
Comment
In an attempt to identify new antibacterial compounds, we have assembled a diverse range
of azide and alkyne coupling partners for the purpose of creating compound libraries using
Cu(I)-mediated azide-alkyne cycloadditions [CuAAC] (Houston et al., 2008; Wilkinson et al.,
2009). Salicylates such as bismuth subsalicylate have been used for many years to treat
diarrhea and other gastrointestinal disorders (Sox & Olson, 1989). We required a core salicylate
scaffold that could be readily transformed into a variety of derivatives. Here, we describe the
synthesis and X-ray crystal structure of 2'-propynyl 2-hy-droxybenzoate (propargyl salicylate)
(I) using our chemoselective method of boric acid-mediated esterification (Houston et
al., 2004; 2007). Borate can activate hydroxycarboxylic acids such as salicylate toward
esterification under mild conditions that are tolerant to acid-labile functional groups such as
alkynes. This ester was previously synthesized by alkylation for the synthesis of cobalt
carbonyl complexes and study of their anti-tumour activity (Jung et al., 1997).
Compound (I) was synthesized cleanly from salicylic acid and propargyl alcohol in 55 % yield
using 10 mol% boric acid in acetonitrile (Levonis et al., 2007) (Fig. 1), and crystallizes from
toluene with two independent molecules in the asymmetric unit (Fig. 2). The ester group
adopts the transoid arrangement (Wilkinson et al., 2006) as stereoelectronic requirements are
met when the carbonyl bifurcates the methylene H atoms (Wiberg & Laidig, 1987). This allows
both p → π and n → σ* overlap from the propargylated oxygen to the carbonyl. The propynyl
groups are co-planar with the 2-hydroxybenzoate; with the intra-molecular O—H···O hydrogen
bond between the phenolic proton and the carbonyl oxygen providing rigidity to the structure
(Table 1). These factors result in the extension of the propynyl group away from the aromatic
core and ensures that the reactivity of the alkyne when using the CuAAC method is not
54
compromised by steric constraints. In the crystal lattice, the propynyl groups form inter-
molecular C—H···O interactions with the phenolic oxygen (Table 1). Supramolecular chains
along the direction of the b axis are found for both molecules with links by weak O1—H1···O7
(molecule A) and C16—H16···O17 (molecule B) inter-molecular interactions (Table 1, Fig. 3).
Experimental
To a stirred solution of salicylic acid (208 mg, 1.5 mmol) and propargyl alcohol (84 mg, 174
mL,3.0 mmol) in acetonitrile (3 ml) was added boric acid (9 mg, 0.15 mmol). The solution was
heated and maintained at reflux for 16 h before concentrating in vacuo. Flash column
chromatography was performed on silica using ethyl acetate as the mobile phase to yield
145 mg (55 %) of (I) as a white solid. This was initially recrystallized from MeOH to furnish
white needles (31 %) for NMR analysis. A second recrystallization from toluene at 0°C
produced single crystals suitable for X-ray diffraction analysis.
1H NMR (CDCl3 300 MHz, 298 K) δ p.p.m. 2.53 (t, J = 2.4 Hz, 1H), 4.91 (s, 2H), 6.87 (ddd, J = 8.1,
7.35, 1.2 Hz, 1H), 6.96 (dd, J = 8.4, 0.9 Hz, 1H), 7.45 (ddd, J = 8.4, 7.2, 1.8 Hz, 1H), 7.85 (dd, J =
7.9, 1.8 Hz, 1H), 10.5 (s, 1H); 13C NMR (CD3OD, 75 MHz, 298 K) δ p.p.m. 53.7, 77.1, 78.3,
113.2, 118.5, 120.4, 130.9, 137.0, 162.9 170.5; MS(ESI-) 175.1 [M—H+].
55
Figure 23 View of the two independent molecules in (I) with the atom numbering scheme.
Displacement ellipsoids for non-H atoms are drawn at the 40% probability level.
Figure 24 Crystal packing in the structure of (I), viewed down the c axis.
Further supplementary materials are included at the end of this thesis.
56
2.6 Conclusions
The work presented in this chapter demonstrates that it is possible in some instances to use
boric acid as a catalyst for the esterification of carbohydrates displaying an -
hydroxycarboxylic acid. This reaction works particularly well with sialic acid, KDN, and quinic
acid. KDO will not form an ester using this method even though it possesses the necessary
-hydroxycarboxylic acid. This reaction was shown to be ineffective when attempting an
esterification using quinic acid and a monosaccharide. A series of salicylate esters were also
produced and crystallographic studies were performed on the product, propargyl salicylate.
2.7 Experimental
2.7.1 Synthesis of Ammonium 3-deoxy-D-manno-oct-2-ulosonate (KDO, 17)
KDO (17) was prepared as according to a literature method.88 D-Arabinose (5.17 g, 34.4
mmol) was combined with oxaloacetic acid (4.54 g, 34.4 mmol) in distilled water (20 mL)
with stirring. The pH of the solution was brought to pH 11 using NaOH solution (~2.0 mL, 10
M). The reaction mixture was stirred for a further 120 minutes before being acidified to pH
2.0 with Amberlite IR120 H acidic resin and finally neutralising with ammonia. Ion exchange
chromatography was performed on the mixture using cG-400 resin. The solution was
loaded and 1 L of neutral water was passed through the column before the product was
eluted with 0.5 M ammonium bicarbonate. The collected eluent was concentrated and
freeze dried before it was dissolved in water (20 mL). Ethanol (15 mL ) was added to this to
O
OHHO
HO
HO
OH
COOH
57
produce a slightly cloudy mixture and ethyl acetate (2 mL) and hexanes (2 mL) were added
before the mixture was filtered through Whatman #40 ashless filter paper. This was
concentrated by vacuum until a thick syrup remained. Three days refrigeration produced
crystals that could be abtained by filtration and washing with ethanol. 1H NMR was
compared with literature values to confirm 17 was formed.95
KDO pyranose: white crystals; yield 42 %; 1H NMR (D2O 300 MHz) 1.69 (dd, 1H, J = 12.6,
5.4 Hz), 1.79 (t, 1H, J = 12.6 Hz), 3.43 (dd, 1H, J = 5.7, 12.0 Hz), 3.60- 3.75 (m, 3H),3.81- 3.91
(m, 2H); 13C NMR (D2O 75 MHz) 33.6, 62.9, 66.1, 66.5, 69.2, 71.5, 96.4, 176.7. MS (ES+)
261.0 (M + Na+).
2.7.2 Attempted synthesis of KDO methyl ester (22)
To a stirred solution of KDO (60 mg, 0.25 mmol) in methanol (anhydrous, 2 mL) was added
boric acid (1.5 mg, 0.02 mmol). The solution was stirred at 500C for 72 hours under a
nitrogen atmosphere before being concentrated by vacuum. 1H NMR spectroscopy of the
reaction crude showed no formation of 22.
2.7.3 Attempted synthesis of KDO phenethyl ester (24)
To a stirred solution of KDO (60mg, 0.25mmol) and 2-phenylethanol (30mg, 0.25mmol) in
acetonitrile (anhydrous, 2mL) was added boric acid (15mg, 0.2mmol). The solution was
stirred for 5 days at room temperature under a nitrogen atmosphere before being
58
concentrated by vacuum. 1H NMR spectroscopy of the reaction crude showed no formation
of 24.
2.7.4 Synthesis of KDN methyl ester (26)
To a stirred solution of KDN (20 mg, 0.08 mmol) in methanol (anhydrous, 2 mL) was added
boric acid (1.5 mg, 0.02 mmol). The solution was stirred for 24 hours under a nitrogen
atmosphere before being concentrated by vacuum. NMR spectroscopy showed the desired
product. Literature reference for 1H NMR96 confirmed formation of 26.
KDN methyl ester: yield > 90 %; 1H NMR (CD3OD 300 MHz) 1.33,1.76, 2.19, 3.58, 3.67,
3.80 (s, 3H, CH3), 3.86, 3.95, 4.02; MS (ES+) 305.0 (M + Na+).
2.7.5 Synthesis of Sialic acid methyl ester (27)
To a stirred solution of sialic acid (200 mg, 0.68mmol) in methanol (2 mL) was added boric
acid (20 mg, 0.33 mmol). The solution was stirred under nitrogen at 500C. After 24 hours
this mixture was concentrated by vacuum and formation of 27 was confirmed by 1H NMR
spectroscopy as compared to literature values.97,98
4
6
5 3
2
O
OH
789
HO
HO
OHH
H OH
RC1
R = NHAc (Neu5Ac)
O
OCH3
4
6
5 3
2
O
OH
789
HO
HO
OHH
H HO
RC1
R = OH
O
OH
59
Sialic acid methyl ester: yield > 90%; 1H NMR (D2O 300 MHz) 2.06 (dd, 1H, J = 12.3, 10.8
Hz), 2.24 (s, 3H), 2.50 (dd, 1H, J = 12.9, 4.5Hz), 3.71 (dd, 1H, J = 4.5, 1.5 Hz), 3.80 (dd, 1H, J =
11.7, 6 Hz), 3.92 (ddd, 1H, J = 9, 6.3, 2.4 Hz), 4.02 (m, 1H), 4.03 (s, 3H), 4.13 (m, 1H), 4.25 (m,
2H); MS (ES+) 346.1 (M + Na+).
2.7.6 Attempted synthesis of glucuronic acid methyl ester (29)
To a stirred solution of glucuronic acid (200 mg, 1.03 mmol) in methanol (anhydrous, 2 mL)
was added boric acid (7 mg, 0.11 mmol). The solution was kept anhydrous under nitrogen
and stirred for 24 hours. Concentration by vacuum before analysis by NMR spectroscopy
showed no formation of 29.
2.7.7 Attempted synthesis of glucuronic acid benzyl ester (30)
To a stirred solution of glucuronic acid (200 mg, 1.03 mmol) and benzyl alcohol (111 mg, 107
L, 1.03 mmol) in acetonitrile (anhydrous, 2 mL) was added boric acid (7 mg, 0.11 mmol).
Stirring was maintained under nitrogen for 24 hours. Concentration by vacuum before
analysis by NMR spectroscopy showed no product formation.
60
2.7.8 Synthesis of quinic acid methyl ester (32)
To a stirred solution of quinic acid (200 mg, 1.04 mmol) in methanol (anhydrous, 2 mL) was
added boric acid (7 mg, 0.11 mmol). The solution was stirred at room temperature for 24
hours. Concentration by vacuum followed by NMR spectroscopy confirmed product 32 as
compared to literature values.99
Quinic acid methyl ester: yield > 90 %; 1H NMR (300 MHz, CD3OD) 1.83 (dd, 1H, H3ax, J =
8.2, 7.6 Hz), 2.05 (m, 3H, H3eq H7ax H7eq), 3.41 (dd, 1H, H5, J = 8.7, 3.3 Hz), 3.72 (s, 3H,
OMe), 3.99 (ddd, 1H, H4, J = 13.5, 7.2, 4.2 Hz), 4.07 (m, 1H, H6); MS (ES+) 228.9 (M + Na+).
2.7.9 Attempted synthesis of quinic acid 2-phenylethanol ester (34)
To a stirred solution of quinic acid (150 mg, 0.78 mmol) and 2-phenylethanol (95 mg, 0.78
mmol) in acetonitrile (anhydrous, 2 mL) was added boric acid (5 mg, 0.08 mmol). The
reaction mixture was stirred for 24 hours under a nitrogen atmosphere before being
concentrated by vacuum. 1H NMR spectroscopy of the reaction crude showed trace
amounts of 34. The product could not be purified by crystallization or flash column
chromatography.
3
45
6
72
HO C1
OH
OH
HO
O
O
61
2.7.10 Attempted synthesis of quinic acid benzyl ester (36)
To a stirred solution of quinic acid (150 mg, 0.78 mmol) and benzyl alcohol (85 mg, 0.78
mmol) in acetonitrile (anhydrous, 2 mL) was added boric acid (5 mg, 0.08 mmol). The
reaction mixture was stirred for 24 hours under a nitrogen atmosphere before being
concentrated by vacuum. 1HNMR spectroscopy of the reaction crude showed no formation
of 36.
2.7.11 Attempted synthesis of galactose mandelic ester (Scheme 10)
To a stirred solution of D-galactose (500 mg, 2.78 mmol) and mandelic acid (422 mg, 2.77
mmol) in acetonitrile (anhydrous, 2 mL) was added boric acid (17 mg, 0.27 mmol). The
solution was maintained under a nitrogen atmosphere for 24 hours. Concentration by
vacuum was followed by NMR spectroscopy of the crude and showed no formation of the
desired product.
2.7.12 Attempted synthesis of 2-deoxy-D-galactose mandelic ester (Scheme 11)
To a stirred solution of 2-Deoxy-D-galactose (50 mg, 0.30 mmol) and mandelic acid (46 mg,
0.30 mmol) in acetonitrile (anhydrous, 2 mL) was combined with boric acid (19 mg, 0.31
mmol). The solution was maintained under a nitrogen atmosphere for 24 hours.
Concentration by vacuum was followed by NMR spectroscopy of the crude and showed no
formation of the desired product.
62
2.7.13 Synthesis of 2-Phenylethyl salicylate (40)
To a stirred solution of salicylic acid (250 mg, 1.81 mmol) and 2-phenylethanol (222 mg, 218
L, 1.81 mmol) in acetonitrile (3 mL) was added boric acid (11 mg, 0.18mmol). The solution
was maintained at reflux for 16 hours before concentrating in vacuo. Flash column
chromatography was performed on silica using ethyl acetate as the mobile phase. IR data
was consistent with literature values for 40.100
Phenethyl-2-hydroxybenzoate: white needles; yield 17.3 %; FTIR: 3255, 2931, 1673, 1614,
1485, 1325, 1300, 1251, 1157, 1090, 756, 701 cm-1; 1H NMR (CDCl3 300 MHz) 3.08 (t, J =
6.9, 2H), 4.54 (t, J = 6.9, 2H), 6.85 (ddd, J = 8.1, 7.1, 1.2 1H), 6.95 (dd, J = 8.4, 0.9 Hz, 1H),
7.21-7.348 (m, 5H), 7.43 (ddd, J = 8.7, 7.1, 1.5 Hz, H5) 7.79 (dd, J = 8.1, 1.6 Hz, 1H, H6); MS
(ES+) 265.0 (M + Na+).
2.7.14 Synthesis of Hexyl-2-hydroxybenzoate (41)
To a stirred solution of salicylic acid (225 mg, 1.61 mmol) and n-hexanol (164 mg, 201 L,
1.61 mmol) in acetonitrile (3 mL) was added boric acid (10 mg, 0.16 mmol). The solution
was maintained at reflux for 16 hours before concentrating in vacuo. Flash column
1
2
OH3
4
5
6
O
O
1
2 OH3
4
5
6
O
O
1'
2'
3'
4'
5'
6'
63
chromatography was performed on silica using ethyl acetate as the mobile phase. Spectral
data was consistent with literature values for 41.101
Hexyl-2-hydroxybenzoate: colourless oil; yield 6.3 %; 1H NMR (CDCl3 300 MHz) 0.88 (t, J =
6.8, 3H, 3x H6’), 1.28-1.44 (m, 3 x CH2, 2x H5’, 2x H4’, 2x H3’), 1.75 (q, J = 6.6 Hz, 2H, 2x H2’),
4.31 (t, J = 6.6, 2H, 2x H1’), 6.85 (ddd, J = 7.8, 7.1, 1.2 Hz, 1H, H3), 6.94 (dd, J = 8.4, 0.9 Hz,
1H, H4), 7.42 (ddd, J = 8.4, 7.2, 1.8 Hz, 1H, H5), 7.81 (dd, J = 7.9, 1.5 Hz, 1H, H6); MS (ES+)
223.0 (M + H+).
2.7.15 Synthesis of Decyl-2-hydroxybenzoate (44)
To a stirred solution of salicylic acid (250 mg, 1.81 mmol) and decyl alcohol (286 mg, 345 L,
1.81 mmol) in acetonitrile (3 mL) was added boric acid (11 mg, 0.18 mmol). The solution
was maintained at reflux for 16 hours before concentrating in vacuo. Flash column
chromatography was performed on silica using ethyl acetate as the mobile phase. Spectral
data was consistent with literature values for 44.102
Decyl-2-hydroxybenzoate: colourless oil; yield 34.6 %; 1H NMR (CDCl3 300 MHz) 0.85 (t, J =
6.9 Hz, 3H, 3x H10’), 1.23-1.58 (m, 7 x CH2, 2x H9’ – 2x H3’), 1.74 (q, J = 6.6 Hz, CH2, 2x H2’),
4.29 (t, J = 6.6 Hz, CH2, 2x H1’), 6.83 (ddd, J = 7.8, 8.1, 1.2 Hz, 1H, H3), 6.93 (dd, J = 8.4, 0.9
Hz, 1H, H4), 7.40 (ddd, J = 8.5, 7.1, 1.8 Hz, 1H, H5), 7.80 (dd, J = 8.1, 1.5 Hz, 1H, H6); MS
(ES+) 301.1 (M + Na+).
1
2 OH3
4
5
6
O
O
1'
2'
3'
4'
5'
6'
7'
8'
9'
10'
64
2.7.16 Synthesis of Prop-2-ynyl 2-hydroxybenzoate (47)103
To a stirred solution of salicylic acid (208 mg, 1.5 mmol) and propargyl alcohol (84 mg, 174
L, 3.0 mmol) in acetonitrile (3 mL) was added boric acid (9 mg, 0.15 mmol). The solution
was maintained at reflux for 16 hours before concentrating in vacuo. Flash column
chromatography was performed on silica using ethyl acetate as the mobile phase.
Prop-2-ynyl 2-hydroxybenzoate (Propargyl salicylate): white needles; yield 55 %; 1H NMR
(CDCl3 300 MHz) 2.53 (t, J = 2.4, 1H, H3’), 4.91 (s, 2H, 2x H1’), 6.87 (ddd, J = 8.1, 7.35, 1.2
Hz, 1H, H3), 6.96 (dd, J = 8.4, 0.9 Hz, 1H, H4), 7.45 (ddd, J = 8.4, 7.2, 1.8 Hz, 1H, H5), 7.85 (dd,
J = 7.9, 1.8 Hz, 1H, H6); 13C NMR (CDCl3 75 MHz) 52.7, 75.6, 75.7, 111.8, 117.7, 119.4,
130.1, 136.2, 161.7, 162.3; MS (ES-) 175.1 (M - H+).
.
1
2 OH3
4
5
6
O
O
1'
2' 3'
65
Chapter 3
The Fluorescence Sensing of
Free Sialic Acid
66
3.1 Free Sialic Acid
Sialic acid can exist in a biological system in numerous forms. Neu5Ac is the most abundant
of the “sialic acids” found in humans, but for example a sialic acid can be part of a
sialoglycoprotein such as the antigen glycophorin C. It can exist as part of one of many
glycoconjugates, with different forms of incorporated “sialic acids” having slight structural
differences from sialic acid itself. In this chapter the focus will be on free sialic acid, this can
be defined as sialic acid (Neu5Ac) that is not bound at any position and is not part of a larger
molecule or macromolecule. Free sialic acid is found in varying concentrations in human
blood, the reference range being 0.5 – 3 mol/L in serum, higher amounts are found in
urine.49 The concentration of free sialic acid measured in blood samples can be a useful
indicator for a number of diseases, including cancerous states in which the increased levels
of sialic acid positively correlate to the degree of metastasis. Since current methods for
detecting sialic acid are lacking in terms of ease of use, detection sensitivity, detection
selectivity, and availability, an approach to detecting sialic acid using a fluorescent
boronolectin could be advantageous.
3.2 Imine Receptor
Azobenzenes, it has been shown, are not particularly fluorescent molecules.104 This is due
to cis-trans isomerism that occurs when they are irradiated, known as photoisomerism.
Since photoisomerism enables azobenzenes to remain fluorescently silent, any means of
reducing or eliminating the photoisomerisation effect should increase fluorescence output.
An example of an azobene 48, has been reported that has a high fluorescence output, this
was shown by the Yamaguchi group in 2006 to be due to a proximal boronic acid causing the
67
fixation of the planar conformation.105 The highly fluorescent azobenzene 48, reported by
the Kawashima group in 2007, owes its exceptional properties to the strongly electron
withdrawing boron that has enhanced electron withdrawing character due to the
fluorobenzene groups.106 This enhances the boron - nitrogen interactions to give a structure
less likely to photoisomerise. The group went on to show that the fluorescence response
can also be produced in the same way by an appropriately functionalised imine 49.107
Prior to the publication of these results we had explored the possibility of an imine-based
fluorescent boronolectin to target free sialic acid. The imine was produced by combining m-
aminophenylboronic acid (50) and o-formylphenylboronic acid (51) in a 1:1 ratio in
methanol to form the imine 52 in solution (Scheme 13). Rather than attempting to isolate
imine 52, it was used in situ due to the reversible nature of imine formation when using
non-anhydrous solvents. It has been shown that imines such as this can even form in water
in a reversible process.108 A yellow colouration in our case was indication of imine
formation. It was thought that this imine would have an appropriate spatial configuration
for bidentate binding to free sialic acid and that the imine would give an enhanced
fluorescent signal due to the additional conjugation included in the molecule by the
inclusion of the nitrogen double bond. The preliminary results, shown below in Figure 25
indicated the desired outcome.
48
49
NN
B
C6F5
C6F5
N
B C6F5
C6F5
68
Scheme 13
Figure 25 Chart displaying relative fluorescence of imine 52 when combined with different
concentrations of monosaccharides
As shown in Figure 25, the readings were taken at excitation wavelength of 365 nm and an
emission wavelength of 445 nm. This bar graph shows the effect of pH on the fluorescence
output of the imine. Relative fluorescence intensity is shown on the y-axis, and the taller
bars are associated with higher carbohydrate concentrations. All of the solutions were
made in a 1:1 ratio of methanol and 100 mM phosphate buffer. Sialic acid (Neu5Ac) was
combined with the sensor system in 1, 10, 20, and 50 mmol concentrations. Although it
appeared that sialic acid displayed a relatively large fluorescence increase when it was
50 51 52
N(HO)2B
(HO)2B
NH2(HO)2B(HO)2B
H
O
+MeOH
69
combined with the imine, showing the desired outcome, this response was shown to be not
due to the presence of the imine receptor. In this assay sialic acid was not tested as a
control without the presence of the imine receptor. Later when sialic acid was tested as a
control without any receptor added, it was found that the same large fluorescence was
observed. This was due to autofluorescence of sialic acid which complicated the
experiment. Although the presence of autofluorescence will occur with a wide variety of
human and plant tissues such as proteins, it is generally considered that pure
monosachharides tend not to display this feature.109 Sialic acid, though, does display this
property and this is commonly used as a method of detection of the carbohydrate in pure
samples such as in HPLC where background fluorescence is minimal. The initial studies were
performed in a 96 well plate format in a plate reader style Perkin Elmer Victor series
florescence reader. This fluorometer did not have adjustable monochromators, rather it
relied on an array of filters. Due to this instrumental limitation an ideal excitation and
emission wavelength could not be tuned to match the receptor being used and
autofluorescence of the sialic acid could not be avoided. From this point on in our
experiments, fluorescence assays were conducted using a Varian Eclipse series fluorometer
with high precision monochromators, the limitation being that a cuvette was used rather
than a 96 well array, which was more laborious. With high precision adjustable
monochromators the autofluorescence of sialic acid could be avoided due to the relatively
narrow excitation wavelength window at which autofluorescence was displayed.
Preliminary results were obtained to evaluate whether the imine bound receptor system
had merit as a sensor for sialic acid.
70
Figure 26 Preliminary results showing fluorescence quenching with the addition of sialic acid in 1:1
methanol/ 100mM aqueous phosphate buffer mixture at pH 6.2 Ex. 295 Em. 365
Initial results, shown in Figure 26 indicated a measurable change in fluorescence when sialic
acid was added to the imine receptor system 52. Shown in Figure 27 is the desired binding
mode, where the receptor could potentially form in situ to give the species 53 shown on the
right.
Figure 27
Experimental results suggested that that imine 52 formed in the phosphate buffer/methanol
media, this was also indicated by a yellow colouration, and upon addition of sialic acid the
fluorescence was quenched. By producing the speculated species 53 it is postulated that
51
50
53
NH2
B(OH)2
B(OH)2
O
H
+
Sialic acid
N
B
BO
O
O
O OHHN
HOO
O
O
HO
HO
71
the now anionic boron cannot participate to give an extended fluorophore in the aniline
system,24 nor can it aid in the fixation of the planar conformation of the imine.105 Issues
with this system arose when other simple monosaccharides such as glucose and mannose
were applied to the receptor system and a similar decrease in fluorescence was observed.
The possibility could not be dismissed that another kind of imine was forming. The
biologically occurring monosaccharides that were tested other than sialic acid, were
aldohexoses and thusly can exist partially in open chain aldehyde forms. Shown in Figure 28
is an example of the imine that might form between m-aminophenylboronic acid and an
aldohexose.
50
54
Figure 28
3.2.1 m-Aminophenylboronic acid as a sensor
As discussed above, initial results were promising when the two components of the receptor
were combined in methanolic aqueous media, a colour change was noted as well as a
measurable fluorescence output. By either including or excluding sialic acid the fluorescent
output could be observed to change significantly. When other monosaccharides were
utilised in place of sialic acid, the fluorescent output was altered to varying degrees,
indicating the receptor produced was not selective. After further examination it was
discovered that similar results could be obtained by using m-aminophenylboronic acid on its
NB
HO
OO
HOOH
OH
NH2
B(OH)2
Hexose
72
own without any o-formylphenylboronic acid present. As various monosaccharides were
titrated into the methanolic aqueous solution of m-aminophenylboronic acid, a fluorescence
decrease could be observed.
The assays described below were conducted using a cuvette to hold the solution while the
change in relative fluorescence could be read using the fluorometer. By first preparing a
1000 M solution of the receptor (in this case m-aminophenylboronic acid) in methanol and
then adding this to the cuvette in a 1:1 dilution using 200 mM aqueous phosphate as the
other component, a 500 M solution of the receptor could be produced. Solutions of the
monosaccharides were produced by adding the same ratio of methanolic receptor solution
and phosphate buffer to give a buffered solution containing a concentration of 200 mM of
the monosaccharide and 500 M of receptor. In this way by titrating the monosaccharide
solution into the cuvette the receptor and buffer concentration could remain constant while
the concentration of the monosaccharide could be incrementally increased. Before
fluorescence readings were taken the resultant solution in each instance was given time to
equilibrate.
The monosaccharides tested, including sialic acid, exist in aqueous media as an equilibrium
between the pyranose/furanose and the aldehyde/ ketone forms. In solution it is thought
that the aldehyde form could react with the primary amine of m-aminophenylboronic acid
to form an imine. Sialic acid showed the strongest binding of the monosaccharides that
were tested to the monoboronate at pH 6.2 (Figure 29), further results showed this not to
73
be the case at pH 7.8 (Figure 30). Glucose was the least responsive to binding to the
receptor, followed by galactose, and finally mannose when binding was performed at pH
6.2. Fructose also showed strong binding to the receptor at this pH as measured by
fluorescence quenching. The more acidic buffer in this case favours boronate binding to -
hydroxycarboxylic acids, as discussed earlier.15 This could give rise to the higher level of
selective response towards sialic acid seen at pH 6.2 as compared to the results obtained at
pH 7.8.
Figure 29 Fluorescence of m-aminophenylboronic acid in 1:1 MeOH – 200mM aq. phosphate
buffer. (Ex. 287, Em. 400) pH 6.2
When the same assay was conducted at pH 7.8, a different picture emerges, as can be seen
in Figure 30. Under more basic conditions such as this, there is no preference for binding to
the -hydroxycarboxylic acid present on sialic acid. All of the monosaccharides tested, with
74
the exception of fructose, produced a similar response with an incremental fluorescence
decrease being observed when the monosaccharide concentration was increased. Fructose
displayed a very strong binding preference towards the monoboronate at this pH, which can
be expected for all monoboronates such as this with the preference being for D-Fructose >
D-Galactose > D-Glucose.17,20,31 As in the assay conducted at pH 6.2, there was the possibility
of the formation of an imine species due to the presence of the primary amine.
Figure 30 Fluorescence of m-aminophenylboronic acid in 1:1 MeOH – 200mM aq. phosphate
buffer. (Ex. 287, Em. 400) pH 7.8
3.2.3 Protection of reactive amine
The problems associated with the reactive amine present in m-aminophenylboronic acid led
us to consider an alternative strategy. One possibility was to modify the amine such that it
would no longer be able to participate in interactions with the substrate being detected by
75
fluorescence sensing. Towards this end a reaction was conducted by fellow student Ms
Moira West, who provided the protected amine compound 55 for carbohydrate binding
assays. The protected amine 55 was obtained from m-aminophenylboronic acid by
treatment with 1,5-dibromopentane. This resulting tertiary amine 55 would prevent imine
formation with carbohydrate aldehydes. The results showed that mannose in particular
displayed less of a fluorescence decrease when added to the receptor while glucose and
galactose also showed less affinity (Figure 31). The lack of selectivity of this receptor
highlights the need for a bidentate/multidentate system for binding to monosaccharides.
The assay results are shown below in Figure 31. This assay was conducted in the same way
as previously described with increasing amounts of a carbohydrate solution being titrated
into a receptor solution while keeping both the receptor concentration and the pH constant.
As higher concentrations of the carbohydrates are reached a decrease is observed in
relative fluorescence (y-axis) which is indicative of the carbohydrate binding to the receptor.
55
N
B(OH)2
76
Figure 31 Fluorescence of compound 55 in 1:1 MeOH – 200mM aq. phosphate buffer. (Ex. 295, Em.
375).
3.3 Covalently bound receptor
The aniline boronic acid type system used in m-aminophenylboronic acid is an example of a
fluorescence “off” site, with binding to a diol producing a decrease in the fluorescent output
of the system. This concept was first demonstrated using anthracene boronic acid binding
to fructose, providing the first example of a boronic acid-based fluorescence sensor for
carbohydrates.24 The boron atom will preferentially exist in its tetrahedral form due binding
to the diol which compresses the O-B-O bond angle. An example of this effect is
demonstrated in work showing the formation of cage like structures that modify the Lewis
acidity of the boron atom.18 The now more Lewis acidic boron can no longer participate
electronically in the extended fluorophore of the system, and a reduction in fluorescence is
observed. This also holds true in other “off” type receptor systems. When “on” and “off”
77
sites are combined in the same molecule, fluorescence quenching is mitigated for most
monosaccharides as they cannot span both sites.
Shown in Scheme 14 is the reduction step in the reductive amination used to produce
receptor compound 56. Sodium borohydride was used in this example as the reducing
agent but sodium cyanoborohydride or sodium triacetoxyborohydride could have
potentially have been used in the same way. The compound could be purified satisfactorily
by first dissolving the concentrated reaction crude in dichloromethane and adding hexanes
dropwise to form a precipitate. The precipitate was collected and dried to give the product
57 in 76 % yield. Even in a covalently bound system such as that shown in receptor 58,
imine formation with aldo sugar monosaccharides readily takes place, which gives rise for
the need of alkylation of the nitrogen, for example an n-methyl modification.
52 58
Scheme 14
Figure 32 shows the relative fluorescence response of receptor 58 at pH 6.2, and
demonstrates that as increasing concentrations monosaccharides were added, a
fluorescence decrease was observed. There was no increase in relative fluorescence output
of the receptor observed with any of the monosaccharides tested. Promisingly, this
receptor showed a strong fluorescence decrease for sialic acid, although there was still
(HO)2B N NH
(HO)2B
NaBH4
MeOH
B(OH)2B(OH)2
78
relatively strong binding to fructose. When comparing these results to the results obtained
when using a monoboronate receptor, there is less fluorescence decrease for mannose at
pH 6.2 than that seen with receptor 55 (Figure 32). It could be the case that there is an
imine formation occurring with the open chain monosaccharides as previously described.
Figure 32 Fluorescence of compound 58 in 1:2 MeOH – 100mM aq. phosphate buffer. (Ex. 275,
Em. 375) pH 6.2
Figure 33 shows the relative fluorescence response of receptor 58, this time at pH 7.8. This
assay displayed that there was a decrease in fluorescence output of the receptor as
increasing concentrations of monosaccharides were added. Due to the higher pH, there was
now no longer a preference for the boronic acids to bind to the -hydroxycarboxylic acid
present on sialic acid. When viewing the results graphically, it can be seen that fructose had
79
a stronger binding result at this pH, and the selectivity towards sialic acid that was observed
at pH 6.2 is no longer apparent.
Figure 33 Fluorescence of compound 58 in 1:2 MeOH – 100mM aq. phosphate buffer. (Ex. 275,
Em. 375) pH 7.8
It is apparent that with the secondary amine available for interaction with aldo sugars, both
boron binding sites cannot be utilised to give selectivity for sialic acid. The solution to this
problem would be to create a bidentate sialic acid receptor that had the same spatial
configuration as 58, without the secondary amine available for aldohexose interaction. In
2009 our group published a paper in Chemical Communications to demonstrate this
effect.110
80
3.4 Chemical Communications
Statement of contribution to co-authored published paper
This chapter includes a co-authored published paper. The bibliographic details of the co-
authored published paper, including all authors, are:
Levonis, Stephan M.; Kiefel, Milton J.; Houston, Todd A. Boronolectin with divergent
fluorescent response specific for free sialic acid. Chemical Communications (Cambridge,
United Kingdom) (2009), (17), 2278-2280.
My contribution to the published paper involved:
The synthesis, spectrocopic characterization and detailed experimental processes of
chemical compounds studied in the article. The completion of all binding assay experiments
with provision of data in presentable graphical form. Writing and proofreading of the
manuscript.
(Signed) _________________________________
Stephan Levonis
82
Stephan M. Levonis, Milton J. Kiefel and Todd A. Houston*
Received (in Cambridge, UK) 14th January 2009, Accepted 13th March 2009
First published as an Advance Article on the web 24th March 2009
DOI: 10.1039/b900836p
A fluorescent boronate receptor with a unique response to free sialic acid has been
developed; this divergent response system may find use in design of other fluorophores to
discriminate between structurally similar analytes.
Boronic acid-based carbohydrate sensors have been developed for a variety of biological
applications from blood glucose monitoring, including glucose-responsive contact lenses, to
‘‘boronolectins’’ for cell-surface carbohydrate identification.111 The reversible esterification
of boronates by carbohydrates in aqueous solution can be sensitively monitored either by
fluorescence quenching, as first demonstrated by Czarnik,24 or through fluorescence signal
increase by elimination of internal quenching mechanisms such as photon-induced electron
transfer (PET), as first reported by Shinkai.25 Recently, James has developed an aniline-based
system (59) that responds to monosaccharide binding with fluorescence signal
enhancement via both locally excited (LE) and twisted internal charge transfer (TICT) states.
32 The binding of sugar diols by the boron effectively turns fluorescence ‘‘on’’, i.e. there is an
increase in fluorescence intensity (I > I0). In order to compete with natural lectins for
carbohydrate identification, boronolectins that are highly selective for particular
83
monosaccharide Ligands must be developed. Sialic acids (e.g. Neu5Ac) are important
carbohydrates ubiquitous to mammalian cell surfaces. Serum levels of sialic acid can be
utilized as a biological marker of a cancerous state.41 Free sialic acid has been shown to be a
superior marker for carcinoma of the uterine cervix and genitourinary malignancies when
compared to total sialic acid and lipid-bound sialic acid measurements.42,112 Improved
fluorescent sensing of free sialic acid within a sample could enable the enhanced detection
of malignancies and other diseases involving aberrant sialic acid levels.44 Elevated free sialic
acid levels in serum can serve as a marker for alcohol abuse as well.48 Most methods to
identify free sialic acid in biological samples require the use of sample purification steps. By
developing a fluorophore that responds uniquely to sialic acid over other biological
components, direct measurement of this monosaccharide in biological samples would
become possible. Here, we report the design of a bis(boronate) receptor (60) for free sialic
acid that contains both an ‘‘on’’ (I > I0) and ‘‘off’’ (I < I0) switch with regard to fluorescent
signal from the aniline moiety upon analyte binding (Fig. 1).
Most common monosaccharides cannot span both boronates and thus should not
significantly modulate the fluorescent output of 60, binding weakly to both the ‘‘on’’ and
‘‘off’’ sites resulting in little change in fluorescence response. The low affinity of hexoses for
monoboronates means simple sugars shouldn’t saturate the receptor (forming a 2 : 1
diester and turning fluorescence ‘‘off’’) until very high concentrations of carbohydrate.
Importantly, this receptor was designed to respond via fluorescence quenching when both
boron sites are esterified as the ‘‘off’’ switch is a boronate conjugated within the aniline
fluorophore. Due to the enhanced affinity of molecules that can serve as divalent ligands,
84
such compounds should cause significant fluorescence quenching even in the presence of
molecules that can bind only one boronate and would potentially cause fluorescence to
increase. Such a receptor system would be unprecedented in the field and an advance
toward improving the sensitivity of determining free sialic acid levels in biological samples
by effectively ‘‘silencing’’ competing sugars.
Sialic acid has two unique structural features that can be targeted with a diboronate
receptor: (1) an extended glycerol side chain and (2) an -hydroxyacid-type arrangement of
the hemiketal at the anomeric position. Previous work on sialic acid–monoboronate
interactions66,68,113,69,15 has shown that boron binding shifts from the glycerol tail to the
anomeric centre as pH decreases.67 The earliest fluorescent, boronate-based sialic acid
receptors of Shinkai and Smith and Taylor used electrostatic interactions to bind the
carboxylate of the sugar.114,115 By using a boronate to target this functional group, receptor
60 should be less affected by competing anions in the biological milieu.
59
Figure 34 Boronolectin designed to bind sialic acid. R = H 58, R = Me 60
NH
(HO)2B
NR
(HO)2B
(HO)2B
O
OH
O
OHAcHN
HO
HO OHOH
OFF
ON
85
This bis(boronate) receptor was readily constructed by sequential reductive aminations of 2-
formylphenylboronic acid first with 3-aminophenylboronic acid–NaBH4 to yield 58116 (76%)
followed by methylation with formaldehyde– NaBH4 (30%) to furnish 60.§ Both 60 and 58
were purified by column chromatography on neutral alumina. Boronate binding of neutral
monosaccharides tends to be favoured at pH values approaching that of the ligand.117,118
Using Shinkai’s glucose receptor,20 we first demonstrated that -hydroxyacid moieties of
tartrate can be targeted with fluorescent bis(boronates)84 and subsequent work by James
has shown that multivalent -hydroxyacid–boron binding is favoured at acidic pH in this
system.119,120 As such, we tested the binding of 60 (33 mM) with various monosaccharides
in both acidic (pH 6.2) and basic (pH 7.8) environments (100 mM aq. Phosphate buffer–
MeOH 2 : 1). As can be seen in Fig. 2, selective response of 60 to sialic acid was best at the
lower pH. Neutral monosaccharides glucose, galactose, mannose and ribose and the anionic
sugar glucuronic acid changed the fluorescence output of 60 very little at the lower pH.
Although ribose had the highest affinity at higher pH, this decreased markedly at acidic pH.
Importantly, the a-methyl sialoside gave a modest decrease but not to the degree of the
free parent sugar. These data support a divalent binding mechanism involving esterification
of both boronates in 60 by sialic acid (Scheme 1). This is further supported by low
resolution, negative ion ESI-MS data (m/z 579). Lactose, a potentially bidentate ligand, failed
to reduce fluorescence of 60 at pH 6.2 (data not shown). Crucially, titration of the receptor
60 with sialic acid in the presence of 50 mM glucose gave closely similar results to titration
with sialic acid alone. Glucose levels in the blood serum of non-diabetic adults are in the
range of 4–7 mM, but can reach 50 mM in diabetics, while free sialic acid levels in the blood
are much lower (0.15–0.2 mM). Thus, divergent response systems like the one described
here may be developed to identify sialic acid levels in unmodified biological samples without
86
enzymic conversion of glucose a priori. The importance of the tertiary amine in 60 is
demonstrated by the fluorescent response of 3 toward the same monosaccharides. While
interaction of 58 with sialic acid still causes the greatest fluorescent decrease at pH 6.2,
other sugars caused significant depression in the fluorescent output of this receptor. This is
likely due to trapping of the acyclic aldehyde form of the sugars through imine formation
with the secondary amine (Scheme 1). This is supported by the fact that m-
aminophenylboronic acid alone gives very similar results to 58. The meta-substitution in
receptor 60 restricts the ability of this molecule to bind simple monosaccharides through
both boronates. Diboronates can readily bind glucose in a divalent sense through diols at
C1/C2 and C4/C6 but this requires a convergent approach of the two boronates.
Figure 35 Fluorescence of compound 60 (33 M) in 2 : 1 100 mM aq. phosphate–MeOH (Ex. 315 nm, Em. 388 nm) pH 7.8
87
Figure 36 Fluorescence of compound 60 (33 M) in 2 : 1 100 mM aq. phosphate–MeOH (Ex. 315 nm, Em. 388 nm) pH 6.2
60 58
88
N
Me
B
BO
O
O
O OHHN
HOO
O
O
HO
Na
m/z = 579
Sialic Acid
multiple bindingmodes possible
Hexose N
B(OH)2
B
HO
OO
HO OH
OH
NMe
(HO)2B
O
O
B
HO
O
HO
OH
OH
NMe
(HO)2B
O
O
B
O
HO
OH
OH
OH
"Off""On"
Hexose Hexose
Figure 37 Possible monosaccharide binding mechanisms of 60 and 58. Only pyranoside forms
shown.
Such an orientation is not available for compound 60. By binding weakly to both the ‘‘on’’
and ‘‘off’’ switches, glucose changes the overall fluorescence output very little (the small
concentrations of each of the two complexes shown in Scheme 1 effectively cancel each
other out: I ≈ I0). Previously, Shinkai reported a diboronate system that will give a CD
response to glucose, but not its methyl glucoside.121 Additionally, we have shown previously
that diboronates can give a fluorescent increase in the presence of one inositol epimer
(chiro), but not the other (myo), an example of ‘‘stereospecific sensing’’.34 Our current work
is complementary and potentially powerful in that we have identified the framework to bind
to a single monosaccharide (sialic acid) selectively and to signal that binding specifically in
preference to other common monosaccharides. The divergent response of the fluorophore
significantly dampens background signals from competing ligands. Only sialic acid causes
60
89
fluorescent quenching of 60 to an extent that it can be titrated. What remains is to extend
conjugation in this system to improve sensitivity of detection. Preorganization of this ditopic
receptor through conformational restriction can improve current affinity.122 Direct
fluorescent measurement of free sialic acid levels in biological fluids will improve the
timeframe of analysis and obviate the need for sample preparation. This divergent response
approach may also find use in other analytical techniques where detection among
structurally similar analytes is required.
Notes and references
§ Data for compound 60: 1H-NMR (300 MHz, MeOD): 2.79 (s, 3H), 4.42 (s, 2H), 6.92 (m,
1H), 7.10 (t, 1H, J = 2.7 Hz), 7.20 (m, 4H), 7.35 (m, 1H), 7.42 (m, 1H); HRMS (ESI) Calcd for
C14H17B2NO4+Na: 306.1309, Found: 306.1302. 1
3.5 Thiophene
Due to the large number of potential fluorophores that could be used to create fluorescence
sensors, the next step in the project was to continue the work by generating further
receptors for sialic acid. Ultimately an improved system would be one that displayed a
greater change in fluorescence at an even lower concentration of analyte. Akin to drug
design, when designing and improving systems such as this there may not be a clear
endpoint to the project, rather a continual evolution. The next iteration in the project was
to produce a system with similar, but slightly altered molecular geometry as well as slightly
altered internal electronic effects.
90
Scheme 15
A contending carbohydrate fluorescence sensor, 62, was produced by reductive amination
of m-aminophenylboronic acid and 2-Formyl-3-thiopheneboronic acid in methanol solvent
using sodium borohydride as the reducing agent. The product had strongly polar properties
such that purification by column chromatography was impossible, crystallisation attempts
were not successful and precipitation form most solvent mixtures did not product a pure
product. This strongly polar compound was found to be soluble in pure water. It was found
that a pure product could be obtained by precipitation from distilled water by adding to it
dropwise a solution of saturated sodium chloride.
Thiophene compounds are of current interest in areas of study where organic chemistry
meets electronic engineering with synthetic oligomers of thoiphenes being used as
semiconductors to produce products such as light emitting transistors.123 Even more
recently thiophene compounds have found usefulness as robust fluorescent indicators with
favourable properties as compared to traditional fluorophores such as fluorocene.124 The
fluorescence response of receptor 62 was examined in a preliminary study and the effects of
adding carbohydrates were observed.
50 61 62
NH2(HO)2B
+
S
B(OH)2
O
HNaBH4
Methanol
NH
S(HO)2B
(HO)2B
91
Figure 38 Fluorescence emmission scan of receptor 62 (Excitiation wavelength = 285 nm)
In Figure 38 is shown the fluorescence emission scan of receptor 62. Shown as marked by
the crosses is the response of the receptor when made to a 33 M solution in a 1:2 mixture
of methanol and pH 6.2 phosphate buffer. Two areas can be seen where there is a
fluorescence emission, the broader peak at approximately emission = 385 nm and a sharper
looking peak further along at approximately emission = 565 nm. When a 50 mM
concentration of glucose was added and the receptor concentration was held constant both
of the peaks diminish in height, indicating a quenching response. Doing the same with sialic
acid (Neu5Ac) causes a much greater response, with near complete quenching of both
peaks. Interestingly, when the same is repeated with fructose, the lower peak is completely
silenced, but the higher peak remains. Due to restraints of time, budget and due to the
92
complicated nature of these results this receptor was not further pursued, future work may
reveal the processes occurring in this system.
3.6 Conclusions
By using know principals of fluorescence modulation we have produced a dual
functionalised boronic acid based fluorescence sensor 60 that shows a high degree of
selectivity for sialic acid. Working through an iterative process of synthesis, testing and
analysis, followed by improvement and modification of successful strategies we arrived at
this goal. By eliminating the possibility of undesirable imine formation with aldohexoses by
methylating at the vulnerable nitrogen, selectivity was achieved.
3.7 Experimental
3.7.1 Example procedure for fluorescence assays, receptor 58
A 100 M stock solution of receptor 58 was produced by dissolving in methanol. A series of
stock solutions were also made of the carbohydrates to be tested. Adding an appropriate
amount of each sugar one part of the methanolic receptor 58 stock solution and two parts
of the 100mM aqueous phosphate buffer solution to give a 1:2 ratio mixture with a 200 mM
concentration of carbohydrate. The carbohydrate mixtures were then allowed 15 minutes
to reach an equilibrium, at this point fluorescence readings could be taken by transferring to
a measurement cuvette to give the relative fluorescence of the 200 mM carbohydrate
mixture. A new mixture was produced comprised of one part receptor solution and 2 parts
phosphate buffer. This solution was then placed into a measurement cuvette and the 200
93
mM carbohydrate solution could be added in incremental amounts with fluorescence
reading taken at each increment. In this way the concentration of the receptor and the
phosphate buffer could remain constant while the carbohydrate concentration could be
gradually increased. The fluorescence was measured on a Varian Eclipse fluorometer
immediately after mixing and at various time points to ensure equilibrium had been
achieved.
3.7.2 Synthesis of 2-((3-boronophenylamino)methyl)boronic acid 58
This compound was prepared according to Yamamoto et al 2004.116 To a stirred solution of
m-Aminophenylboronic acid hemisulphate salt 50 (125 mg, 0.67 mmol) in methanol
(anhydrous, 2 mL) was added 2-formylphenylboronic acid 51 (100 mg, 0.67 mmol) acid. The
solution was kept under an argon atmosphere at room temperature for 24 hours. Sodium
borohydride (28 mg, 0.73 mmol) was slowly added over 15 minutes and the reaction was
stirred at room temperature for a further 24 hours. Saturated sodium chloride solution (25
mL) was added before shaking with ethyl acetate (3 x 30 mL). The ethyl acetate layers were
combined and concentrated by vacuum to give the crude product. Purification could be
achieved by using dichloromethane (1 mL) to dissolve the crude before adding hexanes
dropwise to precipitate the product. 1HNMR was consistent with literature values for 58.125
4''
3''
2''
1''
6''
5''
NH
2
3
4
5
61
(HO)2B
B(OH)2
94
2-((3-boronophenylamino)methyl)boronic acid: white solid; yield 76 %; 1H NMR (300MHz,
MeOD): 4.35 (s, 2H, CH2), 6.74 (m, 2H), 7.15 (m, 2H), 7.32 (m, 4H); MS (ES+) 272.0 (M +
H+).
3.7.3 Synthesis of 2-(((3-boronophenyl)(methyl)amino)methyl)phenylboronic acid 60
Method A
To a stirred solution of the white solid compound 58 (84 mg, 0.31 mmol) and iodomethane
(20 L 47 mg, 0.33 mmol) in acetonitrile (2 mL) was added K2CO3 (200 mg, 1.45 mmol). The
solution was kept under argon at room temperature for 24 hours. The mixture was then
concentrated to dryness before addition of dichloromethane (5 mL) and filtration to remove
solid K2CO3. Concentration to dryness yielded a mixed product, comprising of the
unmethylated, monomethylated and dimethylated amine as evidenced by TLC and mass
spectroscopy. Flash column chromatography on Brockmann grade V acidic alumina was
performed using a mixture of 0.2 % acetic acid, 7 % methanol, 0.8 % water, 25 % ethyl
acetate and 65 % hexanes to give the methylated product 60.
Method B
To a stirred solution of the white solid compound 58 (140 mg, 0.52 mmol) and
paraformaldehyde (40 mg, 1.2 mmol equivalent available formaldehyde) in methanol (2.0
mL) and water (1.0 mL) was added K2CO3 (100 mg, 0.72 mmol). The solution was left to
4''
3''
2''
1''
6''
5''
N 2
3
4
5
61
(HO)2B
B(OH)2
95
dissolve the paraformaldehyde slowly over 24 hours. This mixture was evaporated to
dryness before redissolving in anhydrous methanol (2.0 mL). Sodium borohydride (40 mg,
1.08 mmol) was added over 30 minutes. The mixture was then concentrated to dryness
before addition of dichloromethane (5.0 mL) and filtration to remove K2CO3. Flash column
chromatography on Brockmann grade V acidic alumina was performed using a mixture of
1% acetic acid, 4% methanol, 95% dichloromethane to give the methylated product 60.
2-(((3-boronophenyl)(methyl)amino)methyl)phenylboronic acid: tan solid; yield 21 %
(method A), 32% (method B); 1HNMR (300 MHz, MeOD): 2.79 (s, 3H, NCH3), 4.42 (s, 2H,
CH2), 6.92 (m, 1H), 7.10 (t, 1H, J = 2.7 Hz), 7.20 (m, 4H), 7.35 (m, 1H), 7.42 (m, 1H); 13C NMR
(MeOD 75 MHz) 42.2 (NCH3), 55.0 (CH2), 100.6, 117.8, 122.4, 126.4, 127.7, 128.4, 128.5,
129.0, 129.5, 132.4, 134.3, 144.6; HRMS (ESI) Calcd for C14H17B2NO4 + Na: 306.1309,
Found: 306.1302. 1.
3.7.4 2-F-3-Thiopheneboronic acid receptor
To a stirred solution of 2-Formyl-3-thiopheneboronic acid (95 mg, 0.61 mmol) in methanol
(anhydrous, 1.0 mL) was added m-aminophenylboronic acid hemisulphate salt (114 mg, 0.61
mmol). The solution was stirred for 16 hours. Sodium borohydride (45mg, 1.2 mmol) was
added over 30 minutes with stirring and left for a further 60 minutes. After the removal of
methanol the product could be precipitated by dissolving the crude in water and adding a
small amount of sodium chloride.
6
1
2
3
4
5
NH
2''
3''
4''
5''
S1"
(HO)2B
(HO)2B
96
2-((3-boronophenylamino)methyl)thiophene-3-boronic acid: red solid; yield 32%; 1H NMR
(MeOD 300 MHz) 4.53 (s, 2H, 3-NHCH2), 6.73 (d, J = 7.2, 1H, H4’’), 6.94 (m, 2H, H2, H6’’),
7.08 (d, JH4-H5 = 5.1, 1H, H4) 7.09 (m, 1H, H5’’), 7.17 (d, JH4-H5 = 4.8, 1H, H5); 13C NMR (CDCl3
75 MHz) 45.4 (NHCH2), 117.1, 121.4, 122.1, 123.0, 125.0, 125.7, 129.0, 133.6, 134.3, 137.4;
MS (ES-) 276.4 (M - H+).
97
Chapter 4
Direct and Rapid Amide Bond
Formation
98
4.1 Amide Coupling
Chemical reactions that form amide bonds are valuable tools that are highly useful in many
organic syntheses. It has been observed that roughly one quarter of current
pharmaceuticals contain an amide bond.126 Boronic acids have in the past been used as
catalysts to form amides from a carboxylic acid and an amine. Typically boron reagents
when used as amide bond forming catalysts have had to be used in stoichiometric
amounts.127,128,129,130 The first use of a boron compound in catalytic amounts for an amide
forming reaction was published in 1996 by Yamamoto et al.131 In this work phenylboronic
acids were used as catalysts with 3,4,5-trifluorophenylboronic acid determined to be the
strongest catalyst tested. Typically the yields obtained were greater than 90 % with less
than 24 hours of reaction time.
Figure 39 Proposed catalytic cycle for amidation using a boronic acid catalyst131
ArB(OH)2
O
BO
B
OB
Ar
ArAr1/3
RCO2H
H2O
R OB
Ar
OOH
R N
O
R1
R2
HNR1R2
H2O
RCO2H
R = Ph(CH2)3 Ar = 3,5-(CF3)2C6H2 R1 = Bn, R2 = H
99
It has been proposed that the boronic acid in this case will form a mixed anhydride with the
carboxylic acid, shown on the right side of Figure 39, which is the reactive species. A
method for using boric acid, as opposed to a boronic acid, as a catalyst for this reaction was
published by P. Tang in 2005 where the reaction is demonstrated for a number of different
examples, producing a precipitated amide product that could be obtained by filtration.132 In
these examples benzylamines and cyclic aromatic amines were shown to react using 1 mol
% of boric acid as a catalyst (Scheme 16). Even aniline derivatives with electron withdrawing
groups present that reduce nucleophilicity of the amine reacted with an increased amount
of catalyst.
63 64 65
Scheme 16
132
Building further on the concept of using boronic acids as catalysts, it was shown in 2008 by
D. G. Hall that the ortho-substituted phenylboronic acids, particularly ortho-bromo- and
ortho-iodophenylboronic acid, could be used as waste-free recoverable catalysts for these
reactions at room temperature with high yield. It is stated that the para-substituted boronic
acids have low activity as catalysts, also phenylboronic acids that were substituted at both
ortho positions had poor activity.133 This demonstrates a need for at least one free ortho
position. There is in this case a reverse trend of efficacy in the ortho-halide series (I > Br > Cl
> F) meaning that inductive effects alone are not responsible for the catalytic activity and a
number of factors may be at play. In this chapter an attempt at a novel type of amidation
COOH+
H2N
O
HNB(OH)3, 1 mol %
toluene, reflux
100
reaction will be presented, involving an intramolecular spontaneous mechanism with no
external catalyst, giving surprising results. It is proposed that the reactions proceed
between o-aminophenylboronic acid and an -hydroxycarboxylic acid or the aromatic -
hydroxycarboxylic acid, salicylic acid, by a rapid and spontaneous mechanism involving
initial binding of the boronic acid to the or -hydroxycarboxylic acid.
4.2 Model Reactions
A series of reactions were conducted to assess the efficacy of the reaction.
4.2.1 Salicylic acid and o-Aminophenylbornic acid
A preliminary reaction was conducted by stirring some salicylic acid (42) and o-
aminophenylboronic acid (66) in acetonitrile at room temperature (Scheme 17). No
particular effort was made to keep the reaction anhydrous. After 5 minutes of stirring
visible white precipitate began to form from the very dark solution and continued to
develop over the next 30 minutes. 1H NMR spectroscopy of the precipitate gave indication
that a unique product had formed and this was assumed to be the amide product 67.
Scheme 17
66 42 67
B(OH)2
NH2
+
OH
O
OHAcetonitrile
HN
O OH
(HO)2B
101
The 1H NMR of the crude precipitate (Figure 40) indicates a relatively pure product.
Washing the precipitate with a water/acetonitrile mixture can improve the purity of the
product at the expense of yield. Some traces of salicylic acid starting material can be seen
either side of the doublet of doublets at 7.78 ppm, but given the simplicity of the reaction
the crude product could be considered to be of relatively high purity.
Figure 40 1H NMR (MeOD, 300 MHz) spectra of crude precipitate from the reaction shown in
Scheme 17
13C NMR spectroscopic analysis of the product from the reaction between 66 and 42
confirmed the presence of an amide functionality in compound 67, with a peak present at
163.8 ppm. Further experiments were conducted to evaluate this potentially novel
reaction.
4.2.2 Effects of meta-phenylboronic acid Substitution and Importance of ortho-
substitution
It was found that in contrast to o-aminophenylboronic acid (66), when m-
aminophenylboronic acid (50) was used in its place the reaction formed no product (Scheme
102
18). This gave an indication that the ortho positioning of the substituents is important in the
reaction.
Scheme 18
Another reaction was performed using benzoic acid (68) and o-aminophenylboronic acid
(66). Heating the reaction mixture for 24 hours with stirring at 50°C produced no product as
evidenced by 1HNMR spectroscopy. The reaction shown in Scheme 19 indicates that a
hydroxycarboxylic acid functional group is vital for the reaction to proceed. This supports a
hypothesised reaction mechanism, with boronic acid first binding to the -hydroxycarboxylic
acid before an intramolecular amide bond formation.
Scheme 19
4.2.3 Citric acid and o-Aminophenylboronic acid
Another reaction was conducted using o-aminophenylboronic acid (66) and citric acid (69)
stirred in acetonitrile for one hour with heating at 50°C. The precipitate was filtered and
acetonitrile removed by vacuum. This time 1HNMR and 13CNMR indicated that more
complex interactions may have been at play, and also raised the possibility that the product
50 42
66 68
B(OH)2
+
OH
O
OHAcetonitrile
No Reaction
H2N
B(OH)2
NH2
+
O
OHAcetonitrile
50oC, 24 hours
No Reaction
103
70 may have not existed exactly in the amide configuration shown. This reaction produced
a yield of 72 %.
66 69 70
Scheme 20
The integrated ratios of the 1H NMR spectrum (Figure 41) indicate that a product has
formed in the reaction and it has an appropriate number of hydrogens present. But the
shape and splitting of the multiplet at roughly 2.9 ppm indicates that there could be some
dissimilarity between the CH2 present at position 2 and the CH2 present at position 4,
meaning that there may be some internal bonding interaction.
B(OH)2
NH2
+ HO
O
OH
O OH
O
HO
Acetonitrile
50oC, 1 hour
NH
OHO
O
OH
O
HO
B(OH)2
104
Figure 41 1H NMR (MeOD, 300 MHz) spectrum of 70 (selected regions)
As can be seen in the crude 13CNMR in Figure 42 the dissimilarity between the CH2 groups is
further demonstrated with two clear peaks at 168.8, 172.6 ppm attributed to the attached
carboxyl groups (C1 and C5) as well as two peaks that directly correspond to the CH2 groups
at 38.0, 42.2 (C2 and C4). There are a number of possible configurations that the
molecule could be adopting with internal bonding interactions, some more likely than
others. To determine which configuration this structure might be in, it is useful to look at
other work with boronic acid amides that has been conducted in the past.
105
Figure 42
13CNMR (DMSO, 75 MHz) spectrum of 70 (selected regions)
4.3 Internal interactions in o-Amidophenylboronic acids
There have been attempts previously to make amides that also contain a boronic acid
functional group, but it has been noted historically that there are difficulties in purifying
compounds that have an amine ortho- to a boronic acid on an aromatic ring. In 1960 A. H.
Soloway published a paper on the synthesis of aromatic diboronic acids.134 In the synthesis
106
of 2-acetamidobenzene-1,4-diboronic acid it was noted that on elemental analysis it was in
present the monoanhydride form, possible structures for this proposed were 71 and 72.
71 72
To clarify which of these was the correct configuration of the compound a monoboronate
similar to 71, 2-acetamidobenzene boronic acid, was produced by Soloway and as expected
was shown by elemental analysis to be in the anhydride form. It was noted that it is much
easier to produce the acetamido- derivative of ortho- substituted benzeneboronic acids as
compared to boronic acid substituted at different positions, and it was even suggested that
an intramolecular acylation may be taking place. It was stated that m-aminophenylboronic
acid (50) exists as the free boronic acid rather than the anhydride, this is presumable due to
the anhydride being unable to bridge the extra distance. Also infrared spectra gave weight
to the argument. Compound 71/72 gave a series of sharp peaks at 1639, 1540, and 1494 as
well as broad peaks at 2875, 1600, 1450, 1380, 1220, 1090 and 840 cm-1. It was postulated
that the peak at 1639 was indicative of a carbon nitrogen double bond similar to that seen
in an indolenine system supporting the likelihood of 71 being the observed species, but no
definitive statements could be made due to the complicated nature of the spectra.
In our case the infrared spectrum of 70 was also quite complex. Citric acid can be measured
to have a single peak in the carbonyl region at 1714 cm-1 owing to the carboxylic acid
B(OH)2
B
N
HO O CH3
B(OH)2
B
NHCOCH3
O
3
107
carbonyl stretching. Compound 70 has 3 distinct peaks in this area, at 1732, 1703 and 1638
cm-1. It could be that the peak at 1638 is due to a newly formed carbon nitrogen double
bond cf. Soloway 1960, and the other two distinct peaks indicate that there are two
carbonyl groups in different environments.
To add further evidence to support the formation of a boron – nitrogen bond, Cai and Keana
synthesized a compound with the hypothesised structure 73 in 1991. When the suspected
o-acetamidophenylboronate 73 was analysed by 11BNMR the boron was found to be in the
tetrahedral configuration, the difference between the trigonal and the tetrahedral form
being some 10’s of ppm. For example the 11B resonance peak of compound 73 was
measured to be 4.1 ppm compared to that of compound 74 at 29.2 ppm. To further test
whether the boron was truly in a fixed tetrahedral form they added some sodium hydroxide
to the D2O solution to force the boron to be in the tertrahedral form. The 11B resonance
peak of compound 73 shifted to 2.3 ppm and the 11B resonance peak of compound 74
shifted to 2.5 ppm. The relatively small change in compound 73 indicates that the boron
was originally present in the tetrahedral form.
73 74
In 1994 M. Groziak, A. Ganguly and P. Robinson published a paper on boron heterocycles
that resemble naturally occurring purines.135 In the paper they discuss Soloway’s as well as
HN
B(OH)2
NHCOCH3
H3CO+
-B(OH)2
NHCOCH3
108
Cai and Keana’s previous work giving further valuable information regarding these boron
species. As part of their work, compound 75 was generated as a precursor to produce
another compound. The structure of zwitterionic 75 was confirmed by x-ray
crystallography. The tetracoordinate boron was determined to possess a full negative
charge and bond lengths around the heterocyclic system indicate that the positive charge is
delocalised as shown. Another example of a crystal structure showing a similar boron
heterocycle can be found in Mikhailov’s crystal structure determination of compound 76.136
It is said in this 1994 paper that the structure of 76 could be more accurately represented
with a full B-N bond and a positive charge delocalised as in 75.
Interestingly, as part of this work, one of Soloway’s previous compounds was synthesised
using o-aminophenylboronic acid as a starting material which was prepared in turn by par
hydrogenation of o-nitrophenylboronic acid produced from a low temperature nitration of
in acetic anhydride of commercially available phenylboronic acid.137 Using NMR, elemental
and mass spectral analysis they found that o-aminophenylboronic 66 acid exists in its free
monomeric form 66 as well as an asymmetric didehydro dimer 77. It was found that while
75 is stable in methanol solution or in its solid state, it is prone to hydrolysis that removes
the methyl boronic esters, but the B-N bond remains.
75
76
77
B
NH
N
H
H
H3CO OCH3
B
NH
N
Me
Ph
Bu OCMe
O
B
O
N
B
OH
N
H H
H
109
Figure 43 Possible structures of boron containing heterocycles135
Based on their results and a study of past literature, it was concluded by Groziak et al. that
of the possible structures that this type of boron containing heteroatomic system could
adopt (Figure 43) the structure depicted in 78 gives the most likely form in which these
compounds exist and 83 gives the most accurate representation of the hydrated form.
4.3.1 Proposed Configurations of Reaction Products
Based on this body of research it is fair to conclude that compound 70 is not present
primarily in the amide configuration, but instead as a zwitterionic “boronic imide” type
structure. Electrospray mass spectroscopy agrees with this structure with a peak at 291.7
m/z in negative ion mode. A potential structure of compound 70 is shown below (Figure
44). It can be expected that compound 67 could form a similar structure (Figure 45). To aid
in confirming this, 11B NMR could be conducted as part of future work. X-Ray
crystallography is another analytical method that could be employed to rule out the
possibility of isomeric products.
78
79
80
81
82
83
B
N
X
R
OH
B
N
X
R
OH
B
N
X
R
OH
-
+
B
N
OH
OH
CR
X
B
NCHR
HO OH
X
+
H
-
B
NH
X
R
HO OH
H
110
Figure 44 Proposed structure of compound 70 Figure 45 Proposed structure of compound 67
4.4.1 Mandelic acid and o-Aminophenylboronic acid
Another reaction was conducted using mandelic acid (38) and o-aminophenylboronic acid
(66). It was expected that this reaction would proceed due to mandelic acid being an -
hydroxycarboxylic acid. After stirring in acetonitrile for one hour at 50°C, the reaction was
shown to have formed product 84 in 63 % yield (Scheme 21).
66 38 84
Scheme 21
4.4.2 Malic acid and o-Aminophenylboronic acid
The final reaction in this series was conducted using malic acid (38) and o-
aminophenylboronic acid (66). Again, it was expected that this reaction would proceed due
to malic acid being an -hydroxycarboxylic acid. After stirring in acetonitrile for two hours
at 50°C, the reaction was shown to have generated product 86 in 85 % yield (Scheme 22).
HN
BO
OHO
O
OHHO
O
NH
O
HO
BHO
OH
B(OH)2
NH2
+
Acetonitrile
OH
O
OH
O
HN
OH
B
OHHO50oC, 1 hour
111
Scheme 22
Shown in Table 3 is a summary of the test reactions conducted and their respective yields
Table 3 Summary of reactions
Boronic Amine Carboxylic Acid Product Precipitate Yield
66
38
84
63 % (1 hour)
66
42
67
70 % (1 hour)
66
85
86
85 % (2 hours)
66 85
86
B(OH)2
NH2
B(OH)2
NH2 OH
O
OH
OH
O
OHO
HN
OH
B
OHHO
NH
O
HO
BHO
OH
B(OH)2
NH2
+
Acetonitrile
OH
O
HO
O
OH
B
NH
O OH
OH
O
HOOH
50oC, 2 hours
OH
O
HO
O
OHB
NH
O OH
OH
O
HOOH
B(OH)2
NH2
112
66
69
70
72 % (1 hour)
66
68
No Reaction
0 %
50
42
No Reaction
0 %
To further examine this kind of reaction, a similar reaction was attempted using cystine (87)
and mandelic acid (38) as starting materials with boric acid as a catalyst (Scheme 23). If
such a reaction should proceed it was hypothesised that this could occur via coordination of
the two starting materials by boric acid. After 24 hours heating at 50°C no product had
formed with a mixture of unreacted starting materials indicated by thin layer
chromatography.
87 38
Scheme 23
OH
O
OH
HO
O
OH
O OH
O
HO
O
OH
NH2
SH
O
OH
+
OH
O
OH
Acetonitrile
B(OH)3
No Reaction
50oC, 24 hours
HN
BO
OHO
O
OHHO
OB(OH)2
NH2
B(OH)2
NH2
B(OH)2H2N
113
4.5 Conclusion
The work reported in this chapter describes what may be the fastest direct amide bond
forming reaction utilising a free carboxylic acid and a primary amine as starting materials.
The reaction proceeds rapidly under mild reaction conditions with minimal workup
(filtration) to give product of high purity in good to excellent yield. Given the importance of
amide bond forming reactions and the relative difficulty of amide bond formation in
mainstream organic chemistry, the utility of this simple reaction conducted under mild
reaction conditions becomes apparent. At this stage the configuration in which the
products exist is not clear, the likelihood is that they exist as zwitterionic imine structures.
Further examination using 11BNMR studies may provide insight into the true nature of the
product formed.
4.6 Experimental
The reaction products are illustarted in both their amide and imine configurations - as discussed
earlier in this chapter they may be present more accurately as imine products.
4.6.1 Synthesis of amide/imine product 67
To a stirred solution of salacylic acid (30 mg, 0.22 mmol) in acetonitrile (1.2 mL) was added
2-aminophenylboronic acid (30 mg, 0.22mmol). The solution was maintained at 50 0C for 1
hour before the precipitated product could be obtained by filtration.
6
5
4
3
2
1HN
1'
O
2'3'
4'
5'
6'
OH
(HO)2B6
5
4 3
2
1
NH
1'O
2'3'
4'
5'6'
HO
BHO
OH
114
67: white powder; yield 70 %; 1H NMR (MeOD 300 MHz) 6.81 (ddd, J = 7.8, 7.2, 0.9, 1H),
6.95 (dd, J = 8.4, 0.6, 1H), 7.13-7.27 (m, 3H), 7.37-7.48 (m, 2H), 7.78 (dd, J = 7.8, 1.5, 2H); 13C
NMR (MeOD 75 MHz) 117.1, 119.5, 119.6, 122.8, 128.5, 128.8, 129.2, 130.7, 133.3, 134.7,
136.4, 163.8; MS (ES+) 239.8 (M-H2O + H+) ; HRMS m/z 237.071668 [M + H]+ (calcd for
C13H9B1N1O3(-1), 237.071729).
4.6.2 Test reaction between benzoic acid and o-aminophenylboronic acid
To a stirred solution of benzoic acid (27 mg, 0.22 mmol) in acetonitrile (1.2 mL) was added
o-aminophenylboronic acid (30 mg, 0.22 mmol). The solution was maintained at 50 0C for
24 hours. No product had formed at this stage as evidenced by 1H NMR spectroscopy.
4.6.3 Synthesis of amide/imine product 86
To a stirred solution of malic acid (30 mg, 0.22 mmol) in acetonitrile (1.2 mL) was added 2-
aminophenylboronic acid (30 mg, 0.22 mmol). The solution was maintained at 50 0C for 1
hour before the precipitated product could be obtained by filtration.
86: tan powder; yield 85 %; 1H NMR (MeOD 300 MHz) 2.63 (m, 1H, H2), 2.91 (m, 1H, H2),
4.73 (d, J = 6.0, 1H, H3), 7.15 (m, 1H), 7.30 (m, 2H), 7.54 (m, 1H); 13C NMR (MeOD 75 MHz)
39.8 (C2), 73.8 (C3), 123.0, 128.9, 134.8, 135.1, 163.7, 174.6; MS (ES-) 233.9 (M-H3O+);
HRMS m/z 233.061117 [M + H]+ (calcd for C10H9B1N1O5(-1), 233.061558).
3'
4'
5'
6'
1'
2'
(HO)2BHN 4
3
O
21
OH
OH
O
5'
4'
3' 2'
1'
6'B
NH
4
3
O
2 1
OH
OH
O
HOOH
115
4.6.4 Synthesis of amide/imine product 70
To a stirred solution of citric acid (29 mg, 0.15 mmol) in acetonitrile (1.2 mL) was added 2-
aminophenylboronic acid (20 mg, 0.15 mmol). The solution was maintained at 50 0C for 1
hour before precipitated product could be obtained by filtration.
70: tan powder; yield: 72 %; 1H NMR (MeOD 300 MHz) 2.75 (m, 4H, H2, H4), 7.22 (m, 1H),
7.40 (m, 2H), 7.80 (s, 1H); 13C NMR (MeOD 75 MHz) 38.0 (C2), 42.2 (C4), 74.3 (C3), 122.4,
127.2, 128.9, 133.2, 134.6, 168.8 (C1), 172.6 (C5), 178.6 (NCO); MS (ES-) 291.7 (M+ OH-);
HRMS m/z 291.06636 [M + H]+ (calcd for C12H11B1N1O7(-1), 291.067037).
4.6.5 Synthesis of amide/imine product 84
To a stirred solution of mandelic acid (111 mg, 0.73 mmol) in acetonitrile (1.2 mL) was
added 2-aminophenylboronic acid (10 0 mg, 0.73 mmol). The solution was maintained at
500 C for 2 hours. The solution was filtered and the precipitated product washed with
acetonitrile.
84: grey powder; yield 63 %; 1H NMR (MeOD 300 MHz) 5.40 (s, 1H, CHOH), 7.18 (m, 1H),
7.27-7.37 (m, 5H), 7.48 (m, 2H), 7.60 (m, 1H); 13C NMR (MeOD 75 MHz) 79.3 (CHOH),
1'
2'3'
4'
5'
6'
NH
O
3HO
4
5O
OH
2
1
O
HO
B(OH)2
1'
2'
3'
4'
5'
6'HN
BO
OH
3
4
5
OO
OH21
HO
O
1
2
3 4
5
6
O
HN1'
2'
3'4'
5'
6'
HO
(HO)2B
1
23
4
5
6
O
HN
1'
2'
3'
4'
5'
6'
OH
B
OHHO
116
123.1, 128.0, 128.4, 128.9, 129.0, 129.3, 134.8, 135.1, 140.9, 180.9; MS (ES+) 251.8 (M-H2O
+ H+); HRMS m/z 251.088354 [M + H]+ (calcd for C14H11B1N1O3(-1), 251.087379).
4.6.6 Test reaction between m-aminophenylboronic acid and salicylic acid
To a stirred solution of salacylic acid (15 mg, 0.11 mmol) in acetonitrile (1 mL) was added m-
aminophenylboronic acid hemisulphate salt (20 mg, 0.11 mmol). The solution was
maintained at 50 0C for 2 hours before concentrating by vacuum to give the crude 1HNMR
sample. Starting materials remained unreacted as evidenced by 1HNMR.
4.6.7 Test reaction between cysteine and mandelic acid using boric acid as a catalyst
To a stirred solution of mandelic acid (177 mg, 1.16 mmol) in acetonitrile (2.0 mL) was
added cysteine (200 mg, 1.16 mmol) and boric acid (14 mg, 0.23 mmol) under an argon
atmosphere. After stirring for 24 hours no reaction product was indicated by TLC, only a
mixture of starting materials.
117
Chapter 5
Specific Sensing of KDO
118
5.1 KDO as the Target
KDO is a carbohydrate present in the lipopolysaccharide of gram negative bacteria, its
incorporation into this position is vital to the viability of the bacteria.57,85,86 With strains of
bacteria such as Escherichia coli emerging that show multiple antibiotic resistance,138 the
importance of new generation antibiotics with novel modes of action is at a high. Since KDO
is not present in mammalian cells, it’s conceivable that a systemic agent could be employed
to disrupt KDO biosynthesis or prevent the incorporation of KDO into the
lipopolysaccharide. Whilst the consequences of such an agent to the bacterium are
unknown, the fact that KDO appears to be essential to the viability of Gram-negative
bacteria, suggests that such an agent could have useful antibacterial properties.
88
89
90
17 91
Scheme 24 Biosynthetic steps for the production of KDO in bacteria62
Shown in Scheme 24 are the biosynthetic steps for the production of KDO (17).62 First D-
arabinose 5-phosphate isomerise converts D-ribulose-5-phosphate (88) into D-arabinose 5-
phosphate (89). This is then in turn converted into KDO 8-phosphate (90) by KDO 8-
phosphate synthase before KDO 8-phosphatase acts to convert KDO 8-phosphate (90) to
CH2OH
O
H OH
H OH
CH2OPO32-
1. Ara5P
isomerase OOH
OHHO
OPO32-
OPO32-
COOH PiO
OH
CO2-
HO
HO
OPO32-
HO
Pi
O
OH
CO2-
HO
HO
HO
HO CTP PPi
O
OCMP
CO2-
HO
HO
HO
HO
2. KDO8P
synthase
3. KDO8P
phosphatase
4. CMP-KDO
synthetase
119
KDO (17). It is worth mentioning at this point that in E. coli the gene that encodes for KDO
8-phosphatse has been shown to be non-essential, and in its place a non-specific
phosphatase can be used to produce an “external” source of KDO.139 KDO then is converted
to CMP-KDO (91) using the essential enzyme, CMP-KDO synthase.140 After CMP-KDO is
formed KDO transferase acts to incorporate KDO into lipid A. All Gram-negative bacteria
have a single KDO transferase responsible for the incorporation of KDO into the core region
of their LPS, although the process is different in different bacteria due to varying amounts of
KDO residues present. It can be seen in this pathway that unbound KDO is required for the
production of CMP-KDO and that inhibiting any part of the KDO incorporation process can
be a target for antibacterial agents.
This chapter describes our efforts to develop a novel fluorescence sensor specific for free, or
unbound KDO. By producing a KDO binding agent that shows a change in fluorescence upon
binding, the levels of free KDO can be measured and quantified. If an agent is produced that
has been shown to bind KDO, antibacterial assays could then be conducted to determine if
this kind of KDO binding could have antibacterial effects.
5.2 Receptor Design
A previously reported receptor, 60, has been shown to have the ability to bind sialic acid
with a high degree of selectivity to produce a measurable change in fluorescence.110
Working from this it was theorised that a different geometry would be required to bind
KDO, but structural characteristics that made the previous receptor successful would still be
120
valid. The features of particular importance include the tertiary amine linker and the
bidentate binding with a fluorescence “on” and a fluorescence “off” site. These should be
included into a receptor intended to bind to KDO to enhance selectivity and prevent
unwanted interactions with other monosaccharides. After reviewing available starting
materials and synthetic methods, the following target compound (92) was proposed. It was
thought that this compound could potentially show binding to KDO at both the pseudo--
hydoxycarboxylic acid and the diol side chain. Also the indole system could be fluorescently
advantageous as well as giving added rigidity to the molecule, possibly enhancing binding.
As compared to receptor 60 it can be seen that receptor 92 potentially has a shorter
distance between the boronate binding positions thus favouring the geometry of KDO with
its shorter side chain.
60 92
5.2.1 Receptor and Substrate Relationship: “On” and “Off” Behaviour
The receptor, 92, was hypothesized to behave in a way such that it possesses an “on” type
binding site and an “off” type binding site. When both sites are activated simultaneously, an
“off” type response will occur. This can occur when either a relatively low concentration of
a suitable substrate can span both binding sites and bind in a 1:1 ratio or when such a high
concentration of a less suitable substrate is present that both sites are occupied by two
different molecules of the same substrate, and thus activated in this way. If a substrate
cannot span the receptor and activate both sites simultaneously, it was expected that in the
NR
(HO)2B
(HO)2B
R = Me
N
B(OH)2
(HO)2B
121
solution mixture there would be a random “on” and “off” site activation resulting in little to
no net change in fluorescence until higher concentrations are reached causing activation of
both sites. The need for such a large amount of substrate to cause this indicates that once
one molecule is bound to either site it is sterically unfavoured to bind another, thus the
need for a logarithmic order of concentration increase to produce such results. An example
of this could be seen previously in receptor 60 when examining the response of ribose. No
change in fluorescence was seen from baseline levels until a very high concentration of
ribose was reached, and at this point a slight fluorescence decrease was observed. The
advantage to having both an “on” site and an “off” site in a receptor such as this is apparent
when results indicating a strong divergent response are obtained.
5.2.2 Turning “On” Receptor 92
As shown in Figure 46 when a substrate is bound at the “on” site position, a number of
different interactions will occur in the molecule, overall the interactions combine to give a
measurable fluorescence increase. Here, a brief description of some possible interactions
will be given. Firstly, by binding to the boronic acid the diol or -hydroxycarboxylic acid will
cause the boron to prefer tetrahedral geometry.17 This makes a boron – nitrogen
interaction more likely to occur in this system. It is important to note that this is a distinctly
different interaction to that seen during a photoinduced charge transfer, which would
provide no additional rigidity to the system. The exact nature of the boron – nitrogen
interaction has been debated in other systems, but the interaction has been shown to
eliminate the fluorescence quenching effect of the benzene fluorophore caused by the
proximal nitrogen.32 Although quenching by nitrogen has been reduced, any extension of
122
the benzene fluorophore by the potentially vacant p orbital of the boron will have been
eliminated by the binding event and subsequent tetrahedral configuration of the boron, this
means the benzene fluorophore would be expected to remain fluorescently silent.24 Also,
since the nitrogen in the indole system is now at least partially four coordinate and has
taken on at least a partial positive charge this can be expected to have reduced the overall
fluorescence of the indole system.141 The fluorescent output of this system is hypothesized
to be produced by the active indoleboronic acid fluorophore which benefits from extended
conjugation due to the potentially vacant p orbital of the boron. If the structural rigidity of
the system was enhanced through a boron-nitrogen interaction, similar to the fluorescent
process seen in some boron containing azobenzenes, fluorescent output may increase.106
All things considered, the fact that the phenylboronate moiety cannot act as an electron
“sink” when in its bound, tetrahedral form means that fluorescence will be restored to the
quenched system. This is likely to be the dominant interaction owing to the observed
fluorescence increase upon binding to an appropriate substrate.
Figure 46 Example of “on” site activation
N
B(OH)2
B
OO
substrate
"on"
OH
123
5.2.3 Turning “Off” Receptor 92: Single Site Binding
Shown in Figure 47 is an example of the receptor state during “off” site activation. With the
substrate bound to the boron on the indole system, any extended conjugation aided by the
boron is diminished. Also there will be internal quenching caused by the charge transfer
from the proximal nitrogen into the benzeneboronic acid system. These factors are
responsible for the observed reduction in fluorescence output upon binding to some
substrates.
Figure 47 Example of “off” site activation
5.2.4 Turning “Off” Receptor 92: Dual Site Binding
In Figure 48 an example is shown displaying an overall “off” response cause by activation of
both bonding sites simultaneously. Both of the potentially fluorescent fluorophores have
been silenced by the presence of an anionic boron.
N
B
B(OH)2
"off"O
Osubstra
te
OH
124
Figure 48 Example of both sites being bound, causing an overall “off” response
5.3 Receptor Synthesis
To make a receptor that could potentially bind KDO at two positions simultaneously 6-
indoleboronic acid (93) was first added to a superbase142 solvent mixture comprised of a 1:1
anhydrous mixture of DMSO and DMF saturated with potassium hydroxide. After stirring
for 45 minutes at room temperature, 2-bromomethylphenylboronic acid (94) was added
and the conditions were maintained for a further 45 minutes before dilution with water and
extracting into ethyl acetate. Numerous attempts to purify the product using precipitation,
crystallisation, HPLC and phase extraction failed. Column chromatography on silica gel was
problematic but was eventually performed successfully using alumina as the stationary
phase and ethyl acetate as the mobile phase.
93 94 92
Scheme 25
NH
(HO)2B
+Br
DMSO/DMF
KOH
N
B(OH)2
B(OH)2
(HO)2B
N
B
B
OO
substrate
"off"O
Osubstra
te
OH
OH
125
Shown in Figure 49 is the mechanism for the N-alkylation of the indole starting material.
The superbase mixture first acts to abstract a proton from the nitrogen (indole position 1)
forming an indolyl anion, which can then act as the nucleophile to displace the bromine
leaving group giving product 92. In the product obtained by column chromatography (21%
yield), there was no 3-alkylated contaminant present (see Figure 50), as evidenced by NMR
spectroscopy.
94
Figure 49 Mechanistic rationale for the N-alkylation of indoleboronic acid 93
When ionic salts are used such as sodium or potassium are used, as in our case with
potassium hydroxide, N-alkylation is favoured as compared to the more covalent
compounds such as Grignard-type reagents. Also the solvent choice plays a part with polar,
aprotic solvents favouring N-alkyation more so than non-polar solvents.143
93
92
N 1
2
3
(HO)2B
N
B(OH)2
(HO)2B
H
OH
N(HO)2B
Indolyl Anion
Br
(HO)2B
Br
126
Figure 50 Alkylation is unlikely to occur at indole position 3 due to solvent choice and ionic effects
The difficulty when purifying boronic acids originates from the way in which the boronic acid
moiety behaves when applied to the usual stationary phase materials. Boronic acids are
polar functional groups and when working with a system that has multiple boronic acid
moieties, flash column chromatography may become impossible. For compound 92, silica
was not an appropriate stationary phase, since the high polarity of the solvent system
needed to elute the product meant that the silica itself would be dissolved before the
product would elute. Fortunately it was found that the most deactivated grade of
chromatographic alumina, Brockmann grade V, in its acidic form could be used satisfactorily
as a stationary phase when using neat ethyl acetate as a mobile phase. Based on previous
experience with boronic acids this seemed an unlikely outcome and a much more polar
solvent system was anticipated, even when using a deactivated stationary phase. There is
the possibility that some residual DMSO may have constituted part of the mobile phase,
aiding in purification either by its ability to be a powerful eluent or by further deactivation
of the alumina. A recent article describes an effect, where DMSO can be immobilised on
alumina through its strong hydrogen bonding interactions, which was used as a
93
N 1
2
3
(HO)2B
H
N(HO)2B N(HO)2B
N(HO)2B
RX
R BrOH
127
chromatographic stationary phase for inorganic species.144 By modifying the alumina in this
way retention specificity for a particular metal/matalloid could be altered. Purified 92 was
used in fluorescence assays to determine binding specificity for KDO.
5.4 Fluorescence Evaluation of Receptor Performance
The fluorescence assay conducted to evaluate the binding of receptor 92 to KDO was
performed at pH 7.0, which is also the roughly the optimum pH required for the growth of
E.coli as well as many other pathogenic bacteria. The same methanolic buffer mixture (1:2
MeOH – 200 mM aq. Phosphate buffer) was used as in previous assays with other
receptors.110 A constant concentration of receptor 92 was maintained as the concentration
of substrate was incrementally increased over roughly 3-log-scales. The final substrate
concentration reached was 200 mM while the receptor concentration was held at a
constant 33 M. In case of acidic substrates a neutral salt of the compound was used, for
example sialic acid was used in the form of sialic acid sodium salt and KDO was used in the
form of KDO ammonium salt. 5-deoxy KDO was supplied by Institute of Glycomics PhD
candidate Renee Winzar as the ammonium salt.
The fluorescence results were obtained with receptor 92 in a pH 7.0, 2:1 phosphate buffer
and methanol mixture at an excitation wavelength of 280 nm and an emission wavelength
of 360nm. The carbohydrates that were tested in the assay were sialic acid (Neu5Ac),
glucose, galactose, mannose, fructose, glucuronic acid, KDO, ribose and 5-deoxy KDO along
with the non-carbohydrate, quinic acid. It was observed that a divergent response
128
occurred, with an increase in fluorescence occurring upon the addition of some substrates
and a decrease and eventual silencing occurring with others. This pronounced divergent
response makes this carbohydrate receptor unique among molecules of this kind. At the
extremes, when comparing the response of quinic acid (31) to that of 5-deoxy KDO (95) an
almost symmetrical pattern was seen with the fluorescence output more than doubling with
the addition of quinic acid, before eventual saturation and an observed fluorescence
decrease (vide infra).
31 95
Shown in Figure 51 is a graph illustrating the change in fluorescent output of receptor 92
when increasing amounts of non-acidic carbohydrates were added. It was observed that
generally an increase in fluorescence is seen, except when high concentrations of fructose
are added, upon which a decrease is observed. Initially, at lower concentrations, fructose
causes a relatively sharp rise in fluorescence. The higher concentrations of fructose in this
case may have saturated both binding sites of the receptor. It appeared that initially there
is a preference for the “on” binding site before saturation of both sites causes a
fluorescence decrease.
O
OH
CO2HHO
HO
HOHO
HO
OH
OH
O
OH
129
Figure 51 Fluorescence of receptor 92 in 1:2 MeOH – 200 mM aq. phosphate buffer. (Ex. 280, Em. 360) pH 7.0
Shown in the next graph is the relative fluorescent output of receptor 92 changing with
increasing amounts of acidic carbohydrates, including KDO. It was seen that quinic acid,
used here as the pH neutral sodium salt initially caused an increase in fluorescence at lower
concentrations, followed by an eventual decrease in fluorescence after a higher
concentration was reached. The other acidic carbohydrates all generally caused a decrease
in fluorescence, with KDO and 5-deoxy KDO showing a sharp decrease in fluorescence
beginning at least one log unit below the concentration of sialic acid required to achieve the
same effect.
130
Figure 52 Fluorescence of receptor 92 in 1:2 MeOH – 200 mM aq. phosphate buffer. (Ex. 280, Em.
360) pH 7.0
The advantage to having both an “on” site and an “off” site in a receptor such as this is
apparent when results indicating a strong divergent response are obtained. The
fluorescence increase seen is thought to be due to preferential “on” site activation. If a
substrate shows a preference for one site over the other an initial “on” or “off” type
response might be seen. An example of this could be seen in fructose where a fluorescence
increase is initially seen before a reduction in fluorescence with increasing concentration.
This, along with quinic acid and ribose which show similar behaviour, could indicate that the
“on” site of the receptor is more accessible than the “off” site boronic acid attached directly
to the indole ring. Alternatively, the indole may be providing a hydrophobic surface that has
favourable interactions with carbohydrates once they are bound to the “on” site. Once the
“on” site is occupied, a much higher concentration of substrate is required to also activate
131
the “off” site. Also in these results it can be seen that a much higher concentration of sialic
acid is required to cause a quenching response than is KDO indicating that KDO has
favorable geometry for binding to this receptor as compared to sialic acid.
5.5 Binding Sites on KDO
When the modified carbohydrate, 5-deoxy KDO (95), was assayed with the receptor it was
found that a strong decrease in fluorescence was observed comparable to the decrease in
fluorescence seen with KDO. This illustrates that the 4,5 diol may be non-conductive to
binding to the receptor in a desirable 1:1 ratio. It can be concluded that binding to the
receptor occurs at the -hydroxycarboxylic acid and the 7,8 diol. The slightly stronger
response seen with 5-deoxy KDO may be due to the removal of what is potentially a “decoy”
diol binding site. If the 4,5 diol were bound to one of the binding sites, and the KDO
molecule cannot span both sites when bound in this way, then a reduced response would be
expected for KDO as compared to 5-deoxy KDO (95).
95
Shown in Figure 53 is an example of what 5-deoxy KDO (95) might look like when bound to
receptor 92 in a 1:1 ratio. Much like the example shown in Figure 48 there are many
interactions taking place in this system to reduce the fluorescence output of the system at
when measured at the wavelengths used in the assay. Another interaction which may be
taking place in this 1:1 bound system could be that there is now a further reduction in
O
OH
CO2HHO
HO
HO
132
rigidity introduced via the diol side chain of 5-deoxy KDO, thus adding to the potential for
photoisomerisation of the newly formed molecular species.
Figure 53 Depiction of binding to 5-deoxy KDO producing an “off” response in receptor 92
5.6 Increasing the Acidity: Assay at pH 6.2
The fluorescence of receptor 92 was also assayed at an alternative pH (6.2) when compared
to the data presented previously (conducted at pH 7.0). In conducting this second set of
fluorescence experiments, all other variables (e.g. substrates and concentrations) remained
the same as in the previous experiments. The data from these experiments is shown in
Figure 54. As can be seen, KDO produced a strong response, followed closely by sialic acid.
The key difference is that a divergent response is no longer observed; all of the
carbohydrates eventually produce fluorescence quenching with a high enough
concentration. It has been demonstrated that -hydroxycarboxylic acid binding is favoured
by boronic acids at a lower pH, but in a more acidic medium the tetrahedral configuration of
the boron will be less favourable, this could inhibit the binding of diols.15 Also, it could be
possible that the mildly acidic medium is now preventing the boron – nitrogen interactions
that can occur by attenuating the basicity of the nitrogen, so that if “on” site activation were
to occur a fluorescence increase might not be seen.
OO
C
HO
O O
N
B
B
O
O
HO
HO
133
Figure 54 Fluorescence of receptor 92 in 1:2 MeOH – 200mM aq. phosphate buffer. (Ex. 280, Em.
360) pH 6.2
5.7 Bidentate Boronic Acids as Esterification Catalysts
It was determined in previous work outlined in chapter 2 that KDO will not form a methyl
ester when using boric acid as a catalyst when stirring KDO in methanol with heat. It was
thought that since receptor 92 will bind selectively to KDO (17), presumably at the -
hydroxycarboxylic acid position, receptor 92 could have potential as an esterification
catalyst. KDO was combined with a catalytic amount of receptor 92 and stirred in
anhydrous methanol under inert atmosphere for 3 days with heating at 50°C. Similar to
using boric acid as a catalyst, this reaction did not produce any methylated KDO product.
134
92 17
Scheme 26
A similar lack of reaction was observed when an alternative catalyst, receptor 60 was used
under analogous conditions to those with receptor 92. It is unclear at this stage as to why
KDO is so difficult to esterify under these types of catalytic conditions, or perhaps the
binding of the boronic acid receptor to the 7,8 diol is preferred over the
hydroxycarboxylic acid.
60 17
Scheme 27
5.8 Conclusions
In this chapter a novel boronic acid based fluorescence sensor specific for the bacterial
carbohydrate KDO was presented. Based on the structural geometry of KDO the
components of the system were assembled in a single reaction from commercially available
starting materials to produce a diboronic acid carbohydrate receptor that could be purified
using relatively simple chromatographic techniques. A fluorescence assay demonstrated
N
B(OH)2
(HO)2B
+ O
OH
CO2H
HO
HO
HOHO MeOH
X
12.5 mol%
+ O
OH
CO2H
HO
HO
HOHO MeOH
XN(HO)2B
B(OH)2
10 mol%
135
that the receptor displayed a divergent response when different substrates were added,
depending on the substrate either there was a measurable fluorescence increase, a
fluorescence decrease or no change in fluorescence occurred. KDO was able to be
distinguished from other carbohydrates based on observed strong decrease in the
fluorescence output of the system. The receptor system was shown to be specific to KDO
because it most likely displays a 1:1 binding ratio with this carbohydrate, but in terms of
utility as a fluorescence sensor it could conceivably be used to detect the presence of other
substrates including quinic acid. However, a strong decrease in fluorescence was observed
upon the addition of a low concentration of KDO and 5-deoxy KDO which was not seen with
other carbohydrates.
5.9 Experimental
5.9.1 Synthesis of receptor 92
To a stirred solution of DMF (anhydrous, 0.5 mL) and DMSO (anhydrous, 0.5 mL) was added
KOH (1 pellet, ~200 mg). The solution was stirred under an argon atmosphere for 24 hours
at room temperature. The KOH pellet was removed and 6-Indoleboronic acid (50 mg, 0.31
mmol) was added. The solution was stirred under an argon atmosphere for 45 minutes at
room temperature. 2-bromomethylphenylboronic acid (67 mg, 0.31mmol) was then added.
The conditions were maintained for a further 45 minutes before diluting with water and
5
67
4
54
N 1
2
3
1'
2'3'
4'
5'6'(HO)2B
(HO)2B
136
extracting into ethyl acetate. After concentrating by vacuum, flash column chromatography
was performed using Brockmann grade V acidic alumina with ethyl acetate as the mobile
phase.
1-(2-boronobenzyl)-1H-indole-6-boronic acid tan powder; yield 21 %; 1H NMR (CDCl3 300
MHz) 5.03 (s, 2H, NCH2), 6.43 (d, 1H, JH2-H3 = 3, H3), 7.25(d, 1H, JH2,H3 = 3, H2), 7.33 (m, 2H,
H4’, H6), 7.41 (d, 1H, J = 0.9Hz, H5’), 7.44 (d, 1H, JH4-H5 = 8.1, H4), 7.53 (d, 1H, JH4-H5 = 8.1,
H5), 7.68 (m, 1H, H3’), 7.88 (s, 1H, H7); 13C NMR (CDCl3 75 MHz) 70.9 (NCH2), 100.9 (C3),
110.7 (C7), 117.0, 118.6, 118.9, 119.8, 120.8, 123.9, 125.2, 125.3, 126.7, 128.1, 129.9, 130.5;
MS (ES-) 293.7 (M-H+); HRMS m/z 318.115774 [M - H]+ (calcd for C12H11B1N1O7(-1),
318.115203).
5.9.2 Attempted synthesis of KDO methyl ester boronic acid
To a stirred solution of KDO (105 mg, 0.44 mmol) in methanol (anhydrous, 1.5 mL) was
added boronic acid 60 (12 mg, 0.04 mmol). The solution was stirred under a nitrogen
atmosphere for 24 hours at 500C before being concentrated by vacuum. 1H NMR
spectroscopy of the reaction crude showed no product formation.
5.9.3 Attempted synthesis of KDO methyl ester methylated boronic acid
To a stirred solution of KDO (20 mg, 0.08 mmol) in methanol (anhydrous, 2.0 mL) was added
boronic acid 92 (3 mg, 0.01 mmol) in anhydrous methanol and stirred for 72 hours at 500C
137
under a nitrogen atmosphere before being concentrated by vacuum. 1H NMR spectroscopy
of the reaction crude showed no product formation.
138
Chapter 6
Conclusions and Future Work
139
The work outlined in this thesis has provided further insight into the application and utility
of boron containing compounds in organic chemistry. As well as providing invaluable
results, the work presented in this thesis has highlighted certain areas where further
research is warranted. In the following paragraphs, some possible avenues of investigation
are outlined so that this research may continue on to yield important scientific information.
Chapter 2 described the synthesis of ligands specifically designed to be used in binding
assays to detect certain sugars. Based on the results of a number of different assayed that
were presented in chapters 3 and 5, it can be seen that there are certain binding
interactions that appear to have the potential of being exploited by the use of modified
ligands. Specific binding for sialic acid was obtained, and was shown to be unaffected by the
presence of glucose in solution. Methyl sialoside was shown to interact with the receptor to
a much lesser degree, indicating the involvement of the -hydroxycarboxylic acid moiety.
By testing the receptor against some other modified forms of sialic acid it was shown that
the positions on sialic acid that bind with the receptor could be clearly quantified. Thus
synthesizing further derivatives of sialic acid would be of value. Also, in the work presented
in chapter 5, KDO showed strong binding to the receptor and 5-deoxy KDO displayed an
even stronger response. Generating further structurally modified forms of KDO could be a
means of further examining these interactions. If, for example, the -hydroxycarboxylic
acid moiety was modified and the fluorescence response no longer occurred, it could be
concluded that -hydroxycarboxylic acid binding is crucial. Also the interesting response of
quinic acid causing a strong increase in fluorescence output at pH 7.0 when using receptor
could be further examined by generating modified forms of quinic acid.
140
The assays that were discussed in chapter 3 were run at either pH 7.8 or pH 6.2. For a more
complete picture of carbohydrate binding interactions, performing the assays at a pH closer
to neutral could provide important information because this is where the greatest divergent
response of 92 was seen. To reconfirm the hypothesis that the aniline part of the receptor is
indeed acting as an “on” site, we began to explore the synthesis of the monoboronate
receptor 97 (Scheme 28) with the intention to methylate at the nitrogen thus providing a
better comparison to receptor 60.32 Methylation at the nitrogen of 97 followed by
carbohydrate assays would make an ideal small project and would produce a novel boronic
acid based fluorescence sensor.
60
Scheme 28
Another reaction that could be explored in future could be to introduce a different
functional group at the amine of 58 rather than a methyl group. An attempt to add an
isopropyl functional group to this position could be conducted using 2-bromopropane with a
mild catalytic base such as potassium carbonate (Scheme 29). By producing a novel
carbohydrate receptor such as this with an isopropyl functionality or another functional
group it is possible that selectivity for sialic acid could be further improved due to altered
50 96 97
H2N B(OH)2
+
O
H
HN B(OH)2
NaBH4
MeOH
N(HO)2B
B(OH)2
141
steric or hydrophobic interactions. Although the binding to sialic acid may be diminished by
this added bulk, the exclusion of other molecules may aid overall selectivity. Conceivably a
small library of compounds could be made with modifications at this position.
58 98 99
Scheme 29
One of the key problems encountered during the work presented in chapter 4 was not
purification, as is usually the limiting factor in boronic acid synthesis shown in other
chapters, rather characterization proved to be problematic. Internal interactions within the
molecules being analysed caused difficulty when assigning spectra, and it may be that an
equilibrium exists between the configurations in which the molecule may exist. One future
pathway for further analysis may be 11BNMR studies. Since the boron atom shows a very
discernable difference in terms of observed peak frequency when in its tetrahedral or
trigonal forms, 11BNMR could give insight into this aspect of the state of the molecule.
Another method of characterization that could be of use is x-ray crystallography. As
demonstrated by the propargyl salicylate example shown in chapter 2, x-ray crystallography
can give a very clear picture of internal interactions occurring in a molecule. Something that
could be of concern when using this method, is that the conditions under which the crystal
is grown (aqueous or anhydrous especially) could affect the state in which the compound
crystallizes and may not reflect the state that the compound is in when being observed via
NMR spectroscopy.
HN B(OH)2
K2CO3
DCM
+Br
or acetonitrile
N B(OH)2
B(OH)2B(OH)2
142
The work presented in chapter 5 demonstrates that a compound, 92, was produced that
was shown to bind to KDO via boronic acid functional groups. At this point the question is
raised as to whether this compound could possibly elicit some kind of antibacterial or
bateriostatic effects. The ability of compound 92 to bind to KDO has been demonstrated,
some preliminary antibacterial assays were undertaken by researcher and Griffith University
PhD candidate, Lee Gloyne. MIC values were measured as the compound was tested for
antibacterial activity against the Gram-negative bacteria P. aeruginosa and E. coli as well as
antifungal activity against a strain of Candida. The preliminary antibacterial assays were
inconclusive, but a bacteriostatic effect seemed apparent in preliminary data. More
promisingly, the preliminary assays indicated antifungal activity at 4 M against Candida
albicans. Future research will be required before this data is in a presentable form, but may
bring about the conception of a novel class of antifungal compounds.
92
Overall the work presented in this thesis demonstrates the application and versatility of the
boron atom in modern organic chemistry. The unique behaviour of this metalloid element
is demonstrated to be applied widespread chemical applications, with a focus on its use in
mild and unique catalytic techniques, as well as its use in binding to diols and -
hydroxycarboxylic acids with local electronic consequences. Not only has the work
presented contributed to the broader scientific community, it has produced new pathways
N
B(OH)2
(HO)2B
143
of inquisition that may be followed in future work. By progressing in these new areas of
research, the knowledge of the utility of this unique element may be further expanded.
144
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Appendix
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