1. 1
1. Acknowledgements
There are several people that I would like to thank for their invaluable help during my project.
Firstly, I would like to thank Professor Chris Hunter for his vital guidance and expertise in all
aspects of the research. Secondly, I would like to thank the members of the Hunter group, who
have been more than happy to answer any question that I felt needed answering. I feel special
mentions should go to Hong-mei Sun, Mike Jinks, Rafel Mesquida and Cristina Misuraca who
took the time to explain several difficult, but essential concepts. Finally Iwould like to give
thanks to the University of Sheffield chemistry department, for providing the education and
facilities that made this project possible.
2. 2
2. Abstract
Currently, the imitation of the sophistication and functionality of well-organised supramolecules
systems in Nature is much sought after, as current synthetic machines are not as efficient,
therefore we attempted to analyse a hydrogen bonding interaction between a synthesised
template and product with the hope of forming a system that may self-assemble. The guest
templates and several host diimines were synthesised in good yield and we were able to see
which interactions between them were the strongest, however we were unable to form a system
that showed extensive chelate cooperativity(shown by the calculated effective molarity.) All
calculated effective molarity values were of a similar value and this implies that an increase in
conformational flexibility of our products does not drastically change the effective molarity. This
in turn implies that more flexible design could be a successful strategy for supramolecular
chemists when studied further. Whilst we were unable to attempt the self-assembly of our
product in a dynamic combinatorial library using our templates, this is certainly an area that
could be investigated further.
3. 3
3. Introduction
The objective of this research is to develop synthetic supramolecular approaches to
programmable molecular materials with more precise regulation over chemical structure from
atomic to macroscopic scales. The idea is to replicate the sophistication and functionalityof the
well-organised molecular systems in Nature e.g. nucleic acids, with the currently emerging
synthetic molecular machines. The development of these new molecules will hopefully offer an
efficient synthetic alternative to nucleic acid in encoding, transmitting and expressing
information. A key aspect to this research involves analysing template effects with the option to
extend the study to the formation of a dynamic combinatorial library. Generally, a template is
covalently bonded to a building block, therefore an extra step is required in the reaction scheme
to remove it. In our research we are utilising the reversibility of the hydrogen bond, to
investigate if this non covalent interaction is more efficient in template chemistry.
3.1 What is a template?
A chemical template organizes an assembly of atoms, with respect to one or more geometric loci,
in order to achieve a particular linking of atoms.1
The macroscopic geometry of the reaction is
affected, rather than the intrinsic chemistry which is affected by the reagents involved, so
providing instructions for the formation of a single product from a substrate or substrates which
otherwise may react in other ways.2
In recent years, the development of supramolecular
chemistry has been down to the coalescence of organic and inorganic strands of template
chemistry. This has allowed the utilisation of metal-ligand, hydrogen bonding and π-π
interactions to synthesise larger molecules with greater control.2
Whereas molecular chemistry
essentially deals with the covalentbonding of atoms, supramolecular chemistry is involved in the
4. 4
study of the weaker intermolecular interactions resulting in the association and self-organization
of several components to form larger aggregates3
.
Templates can be separated into having thermodynamic and kinetic effects.
Scheme 1. Kinetic template effects in crown ether synthesis.4
Kinetic templates function under irreversible reaction conditions by stabilizing all transition
states leading to the desired product.2
The longest known and most frequently employed
template syntheses are those based on metal ion chelates, either temporary or permanent.5
A
good example of this process is in crown ether synthesis shown in scheme 1, first developed by
Pedersen by the fortuitous reaction product of an impurity.3
The metal ion template allows the
intramolecular to be favoured over intermolecular reaction, by increasing the proximity of
reactive groups. The speed and specificity of the reaction is hence increased, and the macrocycle
yield compared to the polymer yield is controlled.
5. 5
Scheme 2. Thermodynamic template effects in macrocyclic receptor synthesis.6
Thermodynamic templates function under reversible reaction conditions under thermodynamic
control whereby the template binds most strongly to one of the products and shifts the
equilibrium towards this species. An example of this process is the use of imine exchange to
produce a variety of macrocyclic receptors using metal ion templates in Scheme 2.6
Untemplated
systems containing pyridine dicarboxaldehyde and all three diamines (n=1, 2, 3), produced only
the n=2 product in a 9% yield. However, on addition of the metal template ion, Mg2+
, the
amplification of the n=1 product occurs, being detected in an 86% yield. Larger templates (Ca2+
,
Sr2+
) can be used with the same components but result in poorer yields of the n=1 product.7
3.2 Dynamic Combinatorial Chemistry
Thermodynamic templating is utilised in a concept called dynamic combinatorial chemistry,
which is the chemistry of complex systems under thermodynamic control, i.e. all constituents are
in equilibrium.6
6. 6
Figure 1. (a) Dynamic combinatorial chemistry versus (b) traditional combinatorial chemistry.8
The imine example shown in scheme 2 shows the addition of pyridine dicarboxaldehyde to
several diamines of varying chain length in methanol to produce a dynamic combinatorial library
containing an assortment of macrocyclic and other species in equilibrium. The process involves
rapid interconversion of the library members (potential reactants) by reversible chemical
processes, which can be covalent or non-covalent bonds.6
The method can hence be applied to
identify the most thermodynamically stable structure from a mixture of structures. As a result,
the structures with the most favourable internal interactions will be stabilized and hence formed
preferentially over other library members that lack the stabilization effects.6
A thermodynamic
template can be used to further direct the formation of a specific product by influencing its
structure and geometry within the library, followed by a template removal process.
Figure 2. Linking a mixture of difunctionalised building blocks through a reversible reaction
gives a small dynamic combinatorial library of potential macrocyclic receptors. The free energy
landscape of the system can be seen, whereby the introduction of a template leads to a re-
equilibration in favour of the best receptor. The stability of each macrocycle is reflected in the
depth of its free energy well. 9
7. 7
Figure 2 shows a small dynamic combinatorial library and its free energy landscape. Adding a
template that strongly and selectively binds to one of the equilibrating species, introduces a new
equilibrium in the system, whilst adding an additional free-energy well. If this energy well is
sufficiently deep, the equilibria will shift in the direction of the best host at the expense of unfit
hosts, resulting in an increase in concentration of the selected library member. The resulting
amplification of the ideal host will facilitate identification, and should provide quick and easy
access to large amounts of material.9
This process provides a novel synthetic method, as a
dynamic combinatorial chemist can design a system in which the most successful molecule is
automatically selected and amplified from a pool of potential targets, whereas classically a
molecule has to be designed rather than selected.
There are several factors that are key to the success of this method. Firstly, as previously stated,
the reaction must be reversible, to allow an adequate equilibrium distribution to be produced,
made up of a large number of potential binding patterns before addition of template. If the
reaction is irreversible, the undesired and not necessarily most stable, kineticproduct would
form. Secondly, the library produced must have a shallow energy landscape, to allow for rapid
interconversion between library members. Thirdly, the reaction must be completed under mild
conditions that are compatible with the template and the non-covalent interactions of the
template e.g. at high temperature it is difficult to observe hydrogen bonding between
macromolecules. Finally, it should be possible to turn the reaction off, allowing the isolation and
hence handling of selected members of the library individually.
Dynamic covalent chemistry provides a great opportunity to exploit and explore molecular
recognition in equilibrium mixtures ranging from simple equilibria involving only a handful of
species to large mixtures or dynamic combinatorial libraries.10
Since its discovery in the last 20
years, this method has proven valuable in the identification of unexpected molecules with
remarkable binding properties, in providing effective synthetic routes to complex species and
8. 8
offering insight into how chemical systems respond to external stimuli.11
The field of dynamic
combinatorial chemistry is constantly developing with new applications of the technique
emerging all the time. The process is being used as a way of screening compounds involved in
molecular recognition, utilising the ability of the system to select members with stronger non-
covalent interactions. Presently, the main applications are in the selection of topologically
complex molecules (e.g. catenanes), ligands for proteins and receptors for small molecules. The
use of reversible chemistry to study folded structures has been published on three main classes
of molecules: peptides12
, nucleotides13
, and synthetic polymers14
.
Dynamic combinatorial chemistry also holds potential for catalysis. The first studies have
demonstrated that modestly active supramolecular catalysts can be obtained by screening
dynamic combinatorial libraries for affinity for transition-state analogues, resulting in the
discovery of new catalysts.15
While the catalytic efficiency exhibited by enzymes may well be
beyond our reach, it should at least be possible to develop catalysts that exhibit enantioselectivity
by this method.
Developing ligands for biomolecules is another major application of dynamic combinatorial
chemistry that has been reasonably successful. The ability of a dynamic combinatorial library to
shift its product distribution toward library members that are stabilized through noncovalent
interactions can be extremely useful with systems such as proteins or nucleic acids, where the
exact three-dimensional structure is often unknown, difficult to model, or even strongly
dependent on the ligand bound. At present, disulfide exchange16
and imine exchange17
are the
reactions extensively used in the presence of biomolecules.
9. 9
3.3 Transimination
Transimination is an important reaction in our research as several different diimines were
synthesised and the binding interactions between these diimines and synthesised templates were
measured. Amine – imine exchange of sterically unhindered reactants occur surprisingly quickly
at room temperature.18
The mechanism proceeds by nucleophilic addition to the C=N bond in
situ with proton transfer from the amine NH bond to the imine nitrogen.19
Kinetic data has
shown that this transimination step is faster, even in the absence of a proton or metal catalyst,
than standard amine condensation with a carbonyl.18
This fact then prompted the conversion of
an aldehyde group to an imine group in our research to provide a faster transimination step.
Scheme 3. Transimination.
This reaction could have great potential for implementation in dynamic combinatorial systems
based on the exchange of imine bonds in organic solvents, and under mild conditions.
A typical example, widely explored for dynamic combinatorial chemistry, is the hydrazone
reaction.20
The reaction between a hydrazide and a carbonyl is chemoselective, and the
equilibrium favours the hydrazone in aqueous solution. However its equilibrium kinetics are
slow, so to increase the reaction dynamics, without preventing the reaction equilibrium being
reached, a transimination catalyst is used.21
10. 10
Scheme 4. Hydrazone reaction of hydrazide 1 and glyoxylyl-LYRAG 2 in the absence or
presence of aniline.21
It has been shown that the equilibration kinetics of hydrazone formation and transimination can
be significantly accelerated by using aniline as a nucleophilic catalyst, shown in scheme 4.22
3.4 Cooperativity
Cooperativity is an important property of biological systems23
and is a fundamental concept in
understanding molecular recognition and supramolecular self-assembly.24
Cooperativityarises
from the combination of two or more interactions whereby the new system behaves differently
from what was expected from the individual interactions in solution.24
Cooperativity can be
positive or negative depending on whether the interactions favour or disfavour one another.
Two types of cooperativity are recognised: allosteric and chelate.25
Chelatecooperativity is
straightforward to implement in synthetic supramolecular design, and the multivalent approach
to non-covalent chemistry is therefore beginning to find applications in nanotechnology.26
11. 11
Figure 3. A two-site receptor (AA) that interacts with a divalent ligand (BB.) If [BB]0 >> [AA]0,
then only species within the box are populated.24
Chelate cooperativity is a feature of closed self-assembly structures. As shown by figure 3, when
the ligand is present in excess, we can discount any complexes that involve more than one
receptor. In our case, only four states were present for the receptor; free AA, partially bound 1:1
complex, fully bound cyclic 1:1 complex and 2:1 AA:(BB)2 complex. The relationship between
partially bound and fully bound 1:1 complexes can be shown by equation 1:
1
2
𝐾𝐸𝑀 =
[ 𝑐−𝐴𝐴.𝐵𝐵]
[𝑜−𝐴𝐴.𝐵𝐵]
(1)
where K is Kref, EM is the effective molarity and ½ K EM is the ratio between closed and open
species. Equation 1 gives us an insight into the product distribution between the closed species
that forms two hydrogen bonds, and the open species that forms only one hydrogen bond.
Whilst there are several good methods available for estimating the properties of a single point
hydrogen bond interaction27
, whenthere aremultipleintermolecularcontacts, theparameterwhich
is bestused to quantifythechelatecooperativityassociatedwiththeformation of an intramolecular
12. 12
interaction is the product K EM.28
Here, K is the association constant for the corresponding
intermolecular binding interaction under the same conditions, and EM is the effective molarity.28
When K EM >> 1, the intramolecular process is strongly favoured, and efficient assembly of the
complex will take place. For relatively rigid complexes with good geometric complementarity,
where there are no complications due to changes in conformational flexibility or conformational
strain, the values of EM are of the order 10 M.29
Thus for supramolecular systems, where high
affinity binding sites and highly preorganised scaffolds are the norm, K EM >> 1, and
intramolecular interactions are formed quantitatively. When K EM ≈ 1, there are mixtures of
partiallybound states, aswellas the fullyassembledcomplex, and the behaviourof the system may
be strongly dependent on small changes in conditions. When K EM << 1, there are no
intramolecular interactions and no assembly of the desired complex.28
If we view the entire system as a mixture of partially and full bound states, we can calculate the
EM by the summation of the association constants (shown in figure 3) ignoring any polymer
formation:
𝐾 𝑂𝑏𝑠 = 4𝐾𝑟𝑒𝑓 + 2𝐾𝑟𝑒𝑓
2
𝐸𝑀 (2)
𝐸𝑀 =
𝐾 𝑂𝑏𝑠 − 4𝐾𝑟𝑒𝑓
2(𝐾 𝑟𝑒𝑓 )2 (3)
where EM is effective molarity, Kobs is the experimental association constant and Kref is the
intermolecular association constant.
3.5 Research Outline
The research will begin with the synthesis of the two phosphine oxide templates. Transimination
will be explored and the binding constants for the hydrogen bonding interaction between a large
13. 13
number of synthesised diimines and templates will be analysed, to see if any geometric
complementarity can be seen.
Figure 4. Schematic diagram showing the binding process.
Figure 5. Specific structures that are being targeted in this research. The position where the
templates bind to the nitro phenol building blocks is highlighted, followed by the formation of
the diimine product.
Effective molarity values for these interactions will be determined to see if the partially/ fully
bound species are formed in higher proportion to the polymer. The research may then extend to
14. 14
the formation of a dynamic combinatorial library containing our phosphine oxide template,
imine building blocks (host) and varying length diamine linkers. Ideally, a transimination reaction
will occur within the reaction vessel, and the diimine formed with the highest complementarity
to the template will be selected by the system in equilibrium. This product will then be amplified
and we will see which template selectively binds to a certain length of linker. The compound
with the most complementary chain length to each template would give the highest binding
constant, and hence highest EM.30
Further work would then be done on investigatingthe most
efficient formation of this compound by the use of our templates in a dynamic combinatorial
library.
15. 15
4. Results and Discussion
4.1 Synthesis of Templates
The reaction scheme 5 shows the formation of (di-tert-butyl phosphoryl) methanol (1) from a
reaction between formaldehyde (37 % aq) and tert-butyl chlorophosphine. 1 was formed in a
moderate yield of 41 % which equated to 4.34 g. This yield could be explained by the problems
faced during the purification steps, whereby the crude product did not initially dissolve in hot
hexane (as stated in the literature.) This lead to several hot filtration and recrystallization steps
being performed, which ultimately gave a pure product, but may have contributed to the
moderate yield. Whilst I could have tried to optimise this step, 4.34 g of 1 was enough to
comfortably continue to the next part of the experiment.
Scheme 5 then shows 1 reacting with isophthaloyl dichloride and terephthaloyl dichloride
respectively to synthesise the templates bis((di-tert-butyl phosphoryl) methyl) isophthalate (2)
and bis((di-tert-butyl phosphoryl) methyl) terephthalate(3.) 2 was formed in an excellent yield of
85 %, which equated to 2.75 g, whereas 3 was formed in a more moderate yield of 54 %, which
equated to 1.42 g. Both 2 and 3 produced clean 1
H NMR spectra, confirming their purity, and
the absence of any starting material could be seen by the comparison of the 31
P NMR spectra
with 1 (single phosphorous peak at a different chemical shift.)
17. 17
4.2 Pre-test for ideal host concentration for UV/Vis titrations
The Beer-Lambert law was used to obtain an extinction coefficient, ε of 5856.33 M-1
cm-1
, by
dividing the gradient of the graph in figure 6 by the path length of the plate reader. The path
length value was obtained by dividing the concentration of host closest to an absorbance value
of 0.8 (which was 0.2 mM) by the cross sectional area of the ‘well’ in the UV plate. The optimal
absorbance value for the UV/Vis machine is 0.8, as this is the value at which the signal to noise
ratio is reduced, hence providing results of higher sensitivity. This absorbance value was hence
divided by the calculated extinction coefficient to determine the approximate host concentration
of 0.14 mM. Throughout the research, the host concentration used in any binding study was
therefore around this value.
Figure 6. Graph of host absorbance versus host concentration to obtain an extinction
coefficient for 5-hydroxy, 2-nitrobenzaldehyde (4) in toluene at 298 K.
4.3 Determination of the effect of guest absorbance
In order to determine whether the guest used throughout the research could distort any result by
contributing to the absorbance maxima, a UV/Vis experiment was completed whereby a guest
solution (2) of 50 mM was added to toluene and the absorbance was monitored. The amount of
18. 18
guest added to each well in the plate was known, therefore the guest concentration in each well
could be calculated. Using the Beer-Lambert law, an average extinction coefficient of 8.35 M-
1
cm-1
was calculated from each separate solution analysed. With this calculated value, combined
with the knowledge that the maximum amount of guest solution added to each well in the plate
reader was 152 µL, and the maximum guest concentration used in any binding experiment was
10 mM, the maximum absorbance of the guest solution could be calculated. This absorbance
value was calculated as 0.04, hence proving that the guest solution, at the concentrations used
throughout the research, had minimal effect on the absorbance. This experiment provided
clarity, in that any increase in absorbance could now be confidently attributed to the host and
guest binding.
4.4 Calculation for Approximation of Binding Constant
Using the respective α and β values of the substituents of the interaction and the solvent, the
theoretical binding constant for the interaction could be calculated as shown in equation 2.
Figure 7. Hydrogen bonding interaction between nitro phenol and phosphine oxide substituents
in toluene.
ΔG = - (α – αs)(β – βs) + 6 kJmol-1
(2)
Nitro phenol α = 4.731
Phosphine oxide β = 10.231
Toluene αs = 1.028
βs = 2.228
19. 19
ΔG = -23 kJmol-1
ΔG = - RTlnK therefore K ≈ 1 x 104
M-1
in toluene
This value was therefore used as an approximation for any binding study performed during the
research i.e. if the experimentally determined binding constant was within one order of
magnitude of the theoretically derived binding constant, it could be accepted as a real result. This
binding constant value also falls into the range for determination by UV/Vis titration, therefore
made our decision for us with regards to the selection of characterisation technique.
Toluene was the solvent selected for all binding studies, due to its low polarity, hence reduced
interference with any interaction between host and guest. A more polar solvent e.g. DMSO, may
allow for a higher host concentration in solution by allowing more solid to dissolve, however this
solvent would not make for an efficient solvent system for our research as it is an exceptional
hydrogen bond acceptor (βs = 8.9.) This acceptor ability would cause the solvent-solute
interaction to dominate over any hydrogen bond interactions between host and guest.
4.5 Binding Studies of Template Isomers with Nitro Phenol Aldehyde
The next step in the research was to analyse the binding constants for the hydrogen bonding
interaction between the two templates that were synthesised (2 and 3) and the host 5-hydroxy, 2-
nitrobenzaldehyde (4.) Association constants for the interactions in figure 8 were determined by
UV/vis titrations and fit by a piece of software called the 14AllMaster. The experiment was
performed as a control, in order to obtain a Kref value required for the calculation of the effective
molarity with the diimine linkers in the later sections of the research.
20. 20
Figure 8. 2:1 complex formed between a) 4.2 and b) 4.3
Figure 9. UV/Vis titration data in toluene at 298 K. a) Data for automatic titration of host (4)
with guest (2) and (b) the corresponding fit of the absorbance at 320, 330 and 350 nm to a 2:1
binding isotherm.
Figure 10. UV/Vis titration data in toluene at 298 K. a) Data for automatic titration of host (4)
with guest (3) and (b) the corresponding fit of the absorbance at 320, 330 and 350 nm to a 2:1
binding isotherm.
21. 21
Table 1. Association constants for the formation of 1:1 and 2:1 complexes of 4:2 and 4:3
determined by UV/Vis absorption titrations in toluene at 298K.
The association constants that were experimentally determined in table 1 were an order of
magnitude smaller than the value that was theoreticallycalculated from the respective α and β
values of the constituents involved in the binding interaction (calculation shown in figure 7.)
However, the theoretically calculated association constant utilised α and β values for nitro phenol
and alkyl phosphine oxide. Our system contained a nitro phenol host with an extra aldehyde
constituent, and an alkyl phosphine oxide with an extra ester constituent. These extra functional
groups may explain why the experimental association constant is lower than theoretically
calculated, as the presence of an ester group instead of an alkyl group adjacent to the phosphine
may reduce the hydrogen bond acceptor ability of a phosphine oxide. Similarly the presence of
an aldehyde group on the nitro phenol may reduce the hydrogen bond donating ability of the
phenol, therefore the α and β values taken from the literature may be slightly different to the
substituents involved in our research.
4.6 Imine Synthesis
As previously discussed in the introduction, 5 was synthesised to allow for a faster transimination
step when synthesising the diimines for binding studies at a later stage in the research. 4 and
aniline were combined in benzene and heated overnight using a Dean Stark apparatus. 1
H NMR
analysis was used to see whether the reaction had gone to completion i.e. examining the absence
of aldehyde and the presence of imine peaks. 5 was obtained in an 87 % yield.
Host Guest K Average M-1
K Error M-1
% Error Fitting Ratio
1.90E+03 2.83E+01 1.49 H:G
3.32E+03 8.77E+02 26.41 H2:G
8.13E+03 3.17E+03 38.96 H:G
3.19E+03 1.88E+03 59.06 H2:G
Compound 2
Compound 4
Compound 3
22. 22
Scheme 6.
4.7 Binding Studies of Template Isomers with Imine
The next step in the research was to analyse the binding constants for the hydrogen bonding
interaction between the two templates that were synthesised (2 and 3) and the host 5.
Association constants for the interactions in figure 11 were determined by UV/Vis titrations and
fit by a piece of software called the 14AllMaster. Similarly to the binding interaction between the
templates and nitro phenol aldehyde, this interaction was performed as a control, in order to
obtain a Kref value required for the calculation of the effective molarity.
Figure 11. 2:1 complex formed between a) 5.2 and b) 5.3
23. 23
Figure 12. UV/Vis titration data in toluene at 298 K. a) Data for automatic titration of host (5)
with guest (2) and (b) the corresponding fit of the absorbance at 330 and 350 nm to a 2:1
binding isotherm.
Figure 13. UV/Vis titration data in toluene at 298 K. a) Data for automatic titration of host (5)
with guest (3) and (b) the corresponding fit of the absorbance at 320, 330 and 350 nm to a 2:1
plus non-specific interaction binding isotherm.
Table 2. Association constants for the formation of 1:1 and 2:1 complexes of 5:2 and 5:3
determined by UV/Vis absorption titrations in toluene at 298K.
As shown by the results in tables 1 and 2, the association constants calculated for interactions
between compounds 2/3 with 4/5 are relatively similar. This shows that the system is working
well, as the binding interactions of the two templates with aldehyde and imine hosts when fitted
Host Guest K Average M-1
K Error M-1
% Error Fitting Ratio
8.67E+03 7.68E+02 8.87 H:G
1.09E+03 1.03E+02 9.45 H2:G
1.22E+03 2.50E+02 20.49 H:G
8.96E+03 7.90E+02 8.81 H2:G
Compound 3
Compound 2
Compound 5
24. 24
to a 1:1 or 2:1 binding isotherm are all within the same order of magnitude. The interaction
between 5 and 2 was refitted to a 2:1 + non-specific interaction binding isotherm for the
determination of a more accurate association constant. This fitting took into account a slight
increase in the gradient of the graph in figure 13.b, at higher guest concentration.
4.8 Diimine Syntheses
The various diimines were synthesised by combining 5 with an individual diamine. The reaction
was left overnight and produced each diimine in very high yields. As previously stated in the
introduction, the transimination reaction above proceeded to completion at a quicker rate than
with the aldehyde analogue (4.)
Scheme 7.
4.9 Binding Studies of Templates with Synthesised Diimines
The next step in the research was to analyse the binding constants for the hydrogen bonding
interaction between the two templates that were synthesised (2 and 3) and each of the
synthesised diimine hosts (6-10.) All association constants for the interactions below were
determined by UV/Vis titrations and fit by a piece of software called the 14AllMaster.
25. 25
Figure 14. 1:1 Complex formed between diimines (6-10) and a) 2 and b) 3.
Figure 15. UV/Vis titration data in toluene at 298 K. a) Data for automatic titration of host (6)
with guest (2) and (b) the corresponding fit of the absorbance at 320, 330 and 350 nm to a 1:1
binding isotherm.
Figure 16. UV/Vis titration data in toluene at 298 K. a) Data for manual titration of host (6)
with guest (3) and (b) the corresponding fit of the absorbance at 340, 350 and 420 nm to a 1:1
binding isotherm.
26. 26
Figure 17. UV/Vis titration data in toluene at 298 K. a) Data for automatic titration of host (7)
with guest (2) and (b) the corresponding fit of the absorbance at 330, 340 and 350 nm to a 1:1
binding isotherm.
Figure 18. UV/Vis titration data in toluene at 298 K. a) Data for manual titration of host (7)
with guest (3) and (b) the corresponding fit of the absorbance at 340, 350 and 420 nm to a 1:1
binding isotherm.
Figure 19. UV/Vis titration data in toluene at 298 K. a) Data for automatic titration of host (8)
with guest (2) and (b) the corresponding fit of the absorbance at 340, 350 and 360 nm to a 1:1
binding isotherm.
27. 27
Figure 20. UV/Vis titration data in toluene at 298 K. a) Data for manual titration of host (8)
with guest (3) and (b) the corresponding fit of the absorbance at 340 and 350 nm to a 1:1
binding isotherm.
Figure 21. UV/Vis titration data in toluene at 298 K. a) Data for manual titration of host (9)
with guest (2) and (b) the corresponding fit of the absorbance at 340 and 350 nm to a 1:1
binding isotherm.
Figure 22. UV/Vis titration data in toluene at 298 K. a) Data for automatic titration of host (9)
with guest (3) and (b) the corresponding fit of the absorbance at 330, 340 and 350 nm to a 1:1
binding isotherm.
28. 28
Figure 23. UV/Vis titration data in toluene at 298 K. a) Data for manual titration of host (10)
with guest (2) and (b) the corresponding fit of the absorbance at 340, 350, 410 and 420 nm to a
1:1 binding isotherm.
Figure 24. UV/Vis titration data in toluene at 298 K. a) Data for manual titration of host (10)
with guest (3) and (b) the corresponding fit of the absorbance at 340, 350, 410 and 420 nm to a
1:1 binding isotherm.
Table 3. Association constants for the formation of 1:1 complexes between diimines (6-10) and
2/3, determined by UV/Vis absorption titrations in toluene at 298K. N/A refers to unrepeated
measurements.
Host Guest K Average M-1
K Error M-1
% Error
Compound 2 1.45E+03 1.50E+02 10.33
Compound 3 8.98E+03 N/A N/A
Compound 2 3.75E+04 1.17E+04 31.11
Compound 3 1.64E+04 N/A N/A
Compound 2 8.40E+03 N/A N/A
Compound 3 2.02E+04 N/A N/A
Compound 2 8.17E+03 N/A N/A
Compound 3 3.09E+04 1.26E+04 40.89
Compound 2 9.09E+03 N/A N/A
Compound 3 8.30E+03 N/A N/A
Compound 6
Compound 7
Compound 8
Compound 9
Compound 10
29. 29
Unfortunately, due to time constraints placed on this project, it was not possible to repeat several
of the titrations, therefore there are several without a K error value. Greater importance was
placed on obtaining at least one result for a wider variety of complexes, in order to see if a trend
existed.
Figure 25. The effect of changing the number of carbon atoms in the central chain of our host
compounds on the association constant for the binding interactions with (2) and (3) templates.
From the data, it appears that the most complementary diimine for 2 contains three carbons in
the alkyl chain, whereas the most complementary diimine for 3 contains five carbons in the alkyl
chain. These two systems seem to have the best geometric complementarity, whereby any
complications due to changes in conformational flexibility or conformational strain are at a
minimum. Whilst I would need to repeat the experiments several times before any definitive
statement is made, from initial testing, it seems that the 7-2 and 9-3 hydrogen bonding
interactions are the strongest.
It also appears that, with the exception of the diimine with three carbons in its alkyl chain, the
experimental association constants are greater for 3 than 2, which may be down to a slightly
larger binding pocket in 3.
30. 30
4.10 Calculating the Effective Molarity
By varying both geometric complementarity and conformational flexibility of diimine linker, it
was then possible to quantify these effects on the effective molarity (EM) for the intramolecular
hydrogen bond interaction in the system.
Table 4. Effective molarities (EM/mM) associated with the formation of the intramolecular
hydrogen bond in the closed host and guest complexes at 298 K in toluene.
The EM values shown in table 4 are all extremely low with the blank spaces equating to systems
that do not make a second intramolecular hydrogen bond, as the Kobs values are less than four
times as large as the Kref values. Equation 3 was used to calculate the EM:
𝐸𝑀 =
𝐾 𝑜𝑏𝑠 − 4𝐾𝑟𝑒𝑓
2(𝐾 𝑟𝑒𝑓)2 (3)
where EM is effective molarity, Kobs is the experimental association constant and Kref is the
control association constant.
The low EM values show that there is extremely low cooperativity. There are also two separate
Kref values that have been used for calculating EM, shown by the interactions containing an
aldehyde group in figure 8 and an aliphatic imine group in figure 11. These interactions behave
similarly as the association constants determined are within the same order of magnitude.
However, these control interactions are not strictly correct as they contain an extra aldehyde or
Complex
2.6
2.7
2.8
2.9
2.10
3.6
3.7
3.8
3.9
3.10
3.14
5.33
0.70
3.93
0.10
0.07
0.20
0.84
½ K EM with
aldehyde Kref
1.38
3.87
5.15
8.74
1.15
4
5
6
4.14
0.11
0.07
0.21
2
3
4
5
6
2
Carbon Number in
Diimine Chain
EM (mM) with
aldehyde Kref
EM (mM) with
imine Kref
½ K EM with imine
Kref
3
0.02 0.09
2.36
31. 31
aliphatic imine group to the interaction shown in figure 14. The true Kref value should be
calculated from an interaction between 4-nitrophenol and tri-t-butyl phosphine oxide only.
However, whilst this Kref value may allow for a slightly more accurate calculation of the EM, it
would only minimally affect the result, and it would still be clear that the system did not show
much cooperativity.
For the interactions where a positive EM could be calculated, it was possible to calculatea ½ K
EM value, shown by equation 1:
1
2
𝐾𝐸𝑀 =
[ 𝑐−𝐴𝐴.𝐵𝐵]
[𝑜−𝐴𝐴.𝐵𝐵]
(1)
where K is Kref and ½ K EM is the ratio between closed and open species. For some of the
species, the K EM values are less than one, therefore showing preference for the open system.
However, due to the relatively large Kref values, some of the interactions show considerably
greater preference for the closed species, regardless of the size of the EM for the interaction.
Most noticeably, in the 7-2 interaction, the system shows an approximate ratio of 4:1 for closed:
open, and in the 9-3 interaction, the system shows an approximate ratio of 5:1 for closed: open.
32. 32
5. Conclusion
We have attempted to develop a simple supramolecular model system for investigating the
relationship between linker length and chelate cooperativity by the formation of intramolecular
hydrogen bonds. We successfully synthesised two templates (2 and 3) and several diimines
containing 2-6 carbon atoms in their central chain (6-10), confirmed by several forms of
characterisation. Hydrogen bonding was detected in all of the systems studied, with association
constants for the interactions between the two templates and five diimines recorded in each case.
Whilst further repetition of the titrations would be required to make any definitive statements, it
seemed that the strongest hydrogen bond interactions were between 7-2 and 9-3. These two
systems seem to have the best geometric complementarity, with conformational strain at a
minimum. This statement can be explained by the fact that 2 has a smaller binding pocket than 3
for the diimine to bind based on geometry, therefore 2 will bind most strongly to a smaller
diimine than 3. In our case, 2 bound most strongly to a diimine with three carbon atoms in its
central chain, whereas 3 bound most strongly to a diimine with five carbons in its central chain.
The argument for 3 having a larger binding pocket also agrees with our data whereby, in general,
the association constants for all interactions between 3 with each diimine are larger than the
corresponding interaction between 2 and each diimine. This larger binding pocket may reduce
the steric clash between substituents in our system, allowing for a stronger hydrogen bond
interaction to occur.
The effective molarity for each interaction was then calculated, which was the parameter used to
quantify chelate cooperativity. The EM is defined as the ratio of the intramolecular association
constant for an interaction to the corresponding intermolecular association constant for the same
interaction in a reference system.32
By varying the length of the linker, we were attempting to see
if the value of the EM would drastically change, showing which interaction exhibited the highest
chelate cooperativity. For our system, the association constants vary by, at most, an order of
33. 33
magnitude for all complexes, and the variation in EM is small. The behaviour is very different
from that observed for covalent processes, where there is an increase in EM of several orders of
magnitude for the cyclisation of small rings, and the maximum value is of the order 107
– 1013
M.
Non-covalent EMs values, however, are not as high as the corresponding covalent processes,
which places limitations on the magnitudes of the effects that can be achieved through the use of
chelate cooperativity in supramolecular assembly.
The EM values calculated were extremely low for our system (millimolar), showing that there is
extremely low cooperativity. In many cases the system was mostly forming one hydrogen bond,
as opposed to the desired two. However for the interactions with slightly larger association
constants, and therefore higher EM values, the ratio of open: closed species was greater than one
i.e. ½ K EM was greater than one. Most noticeably, in the 7-2 interaction, the system shows an
approximate ratio of 4:1 for closed: open, and in the 9-3 interaction, the system shows an
approximate ratio of 5:1 for closed: open. Whilst it was not possible to form the fully bound
species for all of the interactions in our system, it was possible for some of the stronger
hydrogen bond interactions.
It was not possible to determine a general relationship between the calculated EM and the
number of rotors i.e. carbon atoms, in the connecting chains, as no real trend existed. However,
as previously stated, the EM values are all similarly small, therefore showing that the decrease in
EM due to the introduction of increased conformational flexibility is less dramatic than might be
expect based on the behaviour of covalent systems.
5.1 Future Work
As stated in the introduction, this research could be extended to the formation of a dynamic
combinatorial library to aid in a more efficient diimine formation. Whilst the formation of a
34. 34
library was attempted during the research, the results were inconclusive, therefore further
importance was placed on finding the strongest host guest interactions by UV/Vis titration. The
dynamic combinatorial library will ideally contain one equivalent of our template, two equivalents
of our nitro phenol building blocks and one equivalent of several diamines of different lengths.
Preferably, several different diimines will be formed in the reaction vessel by a transimination
reaction, before the selection and amplification of the diimine which forms the strongest
hydrogen bonding interaction with our templates.
Potential catalytic effects could also be investigated, whereby our templates could be used to
increase the speed and specificity of the transimination reaction. The template would lower the
∆S‡ for the reaction by increasing the proximity of reactive groups and may even control the
specificity of the reaction by binding substrates in particular orientations. The transimination
reaction in the absence and presence of our templates could be compared to quantify the kinetic
effects of using a template. This reaction could be followed by UV/Vis titration due to the
aromatic substituents in our system being UV active.
35. 35
6. Experimental
Di-tert-butyl phosphoryl methanol
Tert-butyl chlorophosphine (10.5 ml, 10 g) was added dropwise to an aqueous solution of
formaldehyde (100 ml, 37 %) and hydrochloric acid (100 ml, 35 %). The reaction was stirred
under reflux overnight. The reaction was neutralised with sodium hydroxide solution (100 ml, 1
M) and then with sodium hydrogen carbonate solution (50 ml, 1 M). The product was then
extracted from the solution with chloroform (3 x 250 ml) and washed with brine (25 ml). The
solution was then dried with magnesium sulphate and condensed with reduced pressure to give
the crude product. The crude product was purified by recrystallization from hexane at 69 °C and
dried with reduced pressure to give pure (di-tert-butyl phosphoryl) methanol as a white solid
(Compound 1) (4.34 g, 40.8 %);
1
H NMR (250 MHz, CDCl3): δH = 7.28 (s, 1H), 4.05 (s, 2H), 1.31 (d, 18H, J = 13.5);
31
P NMR (101.1 MHz, CDCl3): δp = 59.76 (s, 1P);
13
C NMR (62.8 MHz, CDCl3): δC = 55.4, 54.1, 36.1, 34.3, 26.4;
M.p : 147 – 149 °C;
MS (ES+): m/z (%) = 193.1 (100) [M + H+
];
HRMS (ES+): calcd for C9H22O2P 193.1257, found 193.1343;
FT-IR (ATR): νmax/cm-1
3145, 2960, 2867, 1472, 1367, 1121, 1057.
36. 36
Bis ((di-tert-butyl phosphoryl) methyl) isophthalate
Isophthaloyl chloride (1.27 g, 0.007 mol) was dissolved in dichloromethane (125 ml), protected
by a CaCl2 drying tube. At 0 °C, in a dropping funnel, compound 1 (2.39 g, 0.013 mol) was
dissolved in dichloromethane (25 ml) and triethylamine (2.52 ml, 0.018 mol). The two solutions
were then carefully combined using the dropping funnel, with the resulting colourless solution
stirred overnight, with warming to room temperature. The solution was then washed with 1 %
aqueous sodium hydrogen carbonate (3 x 250 ml) and brine (250 ml). The organic layer was
dried with magnesium sulphate and condensed with reduced pressure to give bis ((di-tert-butyl
phosphoryl) methyl) isophthalate as a pale yellow solid (Compound 2) (2.75 g, 85.4 %);
1
H NMR (250 MHz, CDCl3): δH = 8.71 (s, 1H), 8.26 (d, 2H, J = 8.0), 7.60 (t, 1H, J = 8.0), 4.79
(d, 4H, J = 4.0), 1.39 (d, 36H, J = 14.0);
31
P NMR (101.1 MHz, CDCl3): δp = 55.47 (s, 1P);
13
C NMR (100.6 MHz, CDCl3): δC = 164.96, 134.47, 131.18, 129.89, 129.21, 57.14, 35.44, 26.47;
M.p : 175 – 177 °C;
MS (ES+): m/z (%) = 515 (100) [M + H+
], 532 (10) [M NH4
+
], 537 (20) [M + Na+
], 560 (10)[ M
CH3O2
+
];
HRMS (ES+): calcd for C26H45O6P2 515.2691, found 515.2698;
FT-IR (ATR): νmax/cm-1
2991, 2946, 2901, 2871, 1728, 1471, 1327, 1296, 1227, 1155, 1124, 1076,
819.
37. 37
Bis ((di-tert-butyl phosphoryl) methyl) terephthalate
Terephthaloyl chloride (1.03 g, 0.006 mol) was dissolved in dichloromethane (125 ml), protected
by a CaCl2 drying tube. At 0 °C, in a dropping funnel, compound 1 (1.95 g, 0.010 mol) was
dissolved in dichloromethane (25 ml) and triethylamine (2.05 ml, 0.015 mol). The two solutions
were then carefully combined using the dropping funnel, with the resulting colourless solution
stirred overnight, with warming to room temperature. The solution was then washed with 1 %
aqueous sodium hydrogen carbonate (3 x 250 ml) and brine (250 ml). The organic layer was
dried with magnesium sulphate and condensed with reduced pressure to give bis ((di-tert-butyl
phosphoryl) methyl) terephthalate as a pale yellow solid (Compound 3) (1.42 g, 54.4 %);
1
H NMR (250 MHz, CDCl3): δH = 8.15 (s, 4H), 4.79 (d, 4H, J = 4.5), 1.40 (d, 36 H, J = 15.5);
31
P NMR (101.1 MHz, CDCl3): δp = 55.79 (s, 1P);
13
C NMR (62.8 MHz, CDCl3): δC = 164.86, 133.45, 129.98, 57.91, 34.90, 26.41;
M.p : 176 – 178 °C;
MS (ES+): m/z (%) = 515 (100) [M + H+
], 532 (25) [M NH4
+
], 560 (40)[ M CH3O2
+
];
HRMS (ES+): calcd for C26H45O6P2 515.2691, found 515.2679;
FT-IR (ATR): νmax/cm-1
2988, 2950, 2902, 2874, 1727, 1472, 1327, 1296, 1230, 1147, 1120, 1075,
812.
38. 38
The Determination of Association Constants
Ten wells of a 96 well Hellma quartz plate were filled with 200 µl of solutions containing a range
of concentrations of host in toluene that gave a reasonable curve shape, e.g. 1-20 mM. The
UV/Vis absorbance of the solutions in each vial were monitored using a BMG FLUOstar
Omega plate reader. The vial containing a solution showing an adequate absorbance value of
approximately 0.8 was investigated in further detail to obtain a starting host concentration.
A 5 ml sample of host solution was prepared in spectroscopic grade toluene (dried over calcium
chloride and neutral alumina.) A sample of phosphine oxide guest solution was prepared in
spectroscopic grade solvent, usually in concentrations ranging from 0.6-10 mM. 150 µL of nitro
phenol solution was added to each of 12 wells of a 96 well Hellma quartz plate, and the UV/Vis
absorbance was recorded using a BMG FLUOstar Omega plate reader. The plate reader was
thermostatted at 298 K for all measurements. Increasing aliquots, containing 3, 6 and 9 µL of the
phosphine oxide solution, were added successively to each well containing nitro phenol solution,
and the UV/Vis absorbance was recorded after each addition. Changes in the absorbance values
recorded were fit to a 1:1 or 2:1 binding isotherm, depending on the stoichiometry of the host-
guest complex, using Microsoft Excel to obtain the association constant. Each titration was
repeated once or twice, depending on time constraints, and the experimental error was quoted as
twice the standard deviation at a precision of one significant figure.
Alternatively, association constants were determined using standard manual UV/Vis titration
protocols. Host solutions were prepared at known concentration in spectroscopic grade solvent
and guest solutions were prepared by dissolving the guest solid in a sample of the host stock
solution. This prevented the dilution of the host during the titration. Successive aliquots of the
guest solution were added into the cell, in amounts varying from 10-100 µL, then the UV/Vis
spectrum was recorded. Changes in absorbance were fit to a 1:1 or 2:1 binding isotherm,
depending on the host and guest in question, using a purpose-written software in Microsoft
39. 39
Excel to obtain the association constant. Each titration was repeated once or twice, if time
constraints allowed, and the experimental error was quoted as twice the standard deviation.
40. 40
(E)-4-nitro-3-((phenylimino) methyl) phenol
The nitro phenol aldehyde (Compound 4) (0.30 g, 0.0017 mol) and aniline (0.17 g, 0.0018 mol)
were combined in benzene (12 ml) and heated for 24 hours in a Dean Stark apparatus, producing
(E)-4-nitro-3-((phenylimino) methyl) phenol as a yellow powder (Compound 5) (0.38 g, 87.4 %);
1
H NMR (250 MHz, d6-Acetone): δH = 9.02 (s, 1H), 8.14 (d, 1H, J = 7.5), 7.67 (d, 1H, J = 3.5),
7.49 (d, 1H, J = 8.0), 7.37 (m, 1H), 7.34 (m, 2H), 7.31 (m, 1H), 7.17 (d, 1H, J = 3.0), 7.14 (d, 1H,
J = 3.0);
13
C NMR (62.8 MHz, d6-DMSO): δC = 163.28, 158.10, 151.37, 141.27, 134.23, 129.83, 128.26,
127.14, 121.59, 118.31, 115.39;
M.p : 140 – 142 °C;
MS (ES+): m/z (%) = 243 (100) [M + H+
];
HRMS (ES+): calcd for C13H11N2O3 243.0770, found 243.0762;
FT-IR (ATR): νmax/cm-1
2974, 1731, 1579, 1510, 1327, 1306, 1202, 1075, 861, 840, 764.
41. 41
General Procedure for Diimine Synthesis
Compound 5 (0.48g, 0.002 mol) was added to a diamine (0.001 mol) in acetonitrile (10 ml) and
left to stir overnight. This procedure was completed with diamines containing 2-6 carbon atoms
in the central chain. The acetonitrile was then removed by rotary evaporation to give the separate
diimine products:
49. 49
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