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Department of Organic and Macromolecular Chemistry
Research group Supramolecular Chemistry
Supramolecular Grid Structures For
Hydrogels With Self-Healing Character
Thesis submitted to obtain
the degree of Master of Science in Chemistry by
Ruben Lenaerts
Academic year 2014 - 2015
Promoter: prof. dr. Richard Hoogenboom
Supervisors: Kanykei Ryskulova and Lenny Voorhaar
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Acknowledgments Writing this thesis and thereby finalizing my career as a chemistry student was not possible
without a lot of people, who all have my sincerest thanks.
I would like to thank professor Richard Hoogenboom, for giving me the chance to work in his
research group on my project of choice. I would like to thank him for always being helpful
when the project didn’t go as planned and for always making time to answer my questions
and for his help in writing my thesis.
Gratitude goes to professor Johan Winne, for giving me advice on the synthetic strategy and
making time to look over synthetic problems.
Throughout the year, I have learned a lot and all this would not be possible without my
supervisors Kanykei Ryskulova and Lenny Voorhaar. They learned me how to work in the lab
and helped me writing my thesis. I would like to thank them for always making time when I
had questions.
Furthermore I like to thank everyone in the lab who have helped me with various aspects,
Bart, Maarten and Maarten, Brynn, Joachim, Zhangyao, Gertjan, Victor, Ding-Ying, Mathias,
Glenn, Maji, Kathleen, Jos, Jan, Duchan, Bram and Vincent, who were always willing to spend
time for letting me get to know new practical and theoretical tricks and for creating a great
lab environment.
I would also like to thank all the other master students around in the lab, Mathijs, Jorne,
Lieselot, Jente and Dephine, for the fun moments we experienced.
A special thanks goes to Dries and Roald, for all their help for making my thesis better and all
the fun moments we encountered during our five years as chemistry students.
For all the chromatographic and MS analysis, my sincerest thanks goes to Ir. Jan Goeman, for
all the fast analysis and his willingness for answering my questions. For all their help with NMR
measurements, I would like to thank Tim Courtin, Dieter Buyst and Niels Geudens.
Lastly but no less sincere, I would like to thank my parents for supporting me throughout my
career as a student. Without them, I wouldn’t be able to get a master degree in science. I
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would also like to thank all my friends and my sister for all their support. All of this wouldn’t
be possible without all of you.
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Content 1 Theoretical Part .................................................................................................................. 1
1.1 Aim of the Thesis ......................................................................................................... 1
1.2 Supramolecular Grid structures .................................................................................. 4
1.2.1 Supramolecular Interactions by Metal-Ligand Interactions ................................ 4
1.2.2 Supramolecular Metallogrids ............................................................................... 8
1.2.3 Supramolecular Grids in Solution ....................................................................... 10
1.3 Supramolecular Hydrogels ........................................................................................ 13
1.3.1 Hydrogels ............................................................................................................ 14
1.3.2 Self-Healing Hydrogels ....................................................................................... 15
1.3.3 Applications of Self-Healing Hydrogels .............................................................. 16
1.4 Supramolecular Grids in Polymers and Gels ............................................................. 18
1.5 Supramolecular Metal-Ligand Interactions in Hydrogels .......................................... 20
1.6 Poly(2-oxazoline)s ...................................................................................................... 21
2 Results and Discussion ..................................................................................................... 25
2.1 Synthesis Route ......................................................................................................... 25
2.2 Ligand Synthesis......................................................................................................... 26
2.2.1 Synthesis of 2-Bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3 ........................... 26
2.2.2 Synthesis of 2-[(Triisopropylsilyl)ethynyl]-6-(tributylstannyl)pyridine 4 ........... 31
2.2.3 Stille Coupling Reactions .................................................................................... 34
2.2.4 Synthesis of 4,6-Bis[4’-(ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 7A ................. 38
2.3 Polymer and Oligomer Synthesis ............................................................................... 40
2.3.1 Synthesis of 2-(2-(2-Methoxyethoxy)ethoxy)ethyl Azide 13 ............................. 40
2.3.2 Synthesis of Poly-α-methyl-ω-azido-(2-ethyl-2-oxazoline) ................................ 41
2.4 Cu(I)-Catalyzed Azide-Alkyne Cycloadditions ............................................................ 46
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3 Conclusion and Outlook ................................................................................................... 49
4 Experimental .................................................................................................................... 53
4.1 Materials and Equipment .......................................................................................... 53
4.2 Ligand Synthesis......................................................................................................... 55
4.2.1 2-Bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3, PdCl2(PPh3)2 Catalyst,
Purification by Column Chromatography ......................................................................... 55
4.2.2 2-Bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3, Pd(PPh3)4 Catalyst, Purification
by Column Chromatography ............................................................................................ 56
4.2.3 2-Bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3, Purification by Kugelrohr ..... 57
4.2.4 2-[(Triisopropylsilyl)ethynyl]-6-(tributylstannyl)pyridine 4 ............................... 58
4.2.5 2-[(Triisopropylsilyl)ethynyl]-6-(deuterio)pyridine Test Reaction ..................... 58
4.2.6 2-[(Triisopropylsilyl)ethynyl]-6-(tributylstannyl)pyridine 4 ............................... 59
4.2.7 Stille Coupling Diiodopyrimidine 6 ..................................................................... 59
4.2.8 4,6-Bis[4’-((triisopropylsilyl)ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 6A with
PdCl2(PPh3)2 ...................................................................................................................... 60
4.2.9 4,6-Bis[4’-((triisopropylsilyl)ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 6A with
Tetrakis ............................................................................................................................ 60
4.2.10 4,6-Bis[4’-(ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 7A ..................................... 61
4.3 Polymer and Oligomer Synthesis ............................................................................... 62
4.3.1 2-(2-(2-Methoxyethoxy)ethoxy)ethyl Azide ....................................................... 62
4.3.2 Poly-α-methyl-ω-azido-(2-ethyl-2-oxazoline) .................................................... 63
4.4 Cu(I)-Catalyzed Azide-Alkyne Cycloadditions ............................................................ 65
4.4.1 4,6-Bis[4’-((2-(2-(2-Methoxyethoxy)ethoxy)ethyl)-1”,2”,3”-triazol-4”-yl)pyrid-2’-
yl]-2-phenylpyrimidine 8A-TEG ........................................................................................ 65
5 Bibliography ...................................................................................................................... 67
6 Scientific Article ................................................................................................................ 75
7 Dutch Summary ................................................................................................................ 87
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8 Supporting Info ................................................................................................................. 89
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Abbreviations Asym Asymmetric Bu3SnCl Tributyltin chloride CuAAC Cu(I)-Catalyzed Azide-Alkyne Cycloaddition DCM Dichloromethane DMA N,N-Dimethylacetamide DMAP 4-Dimethylaminopyridine DPP 3,6-Di(2-pyridyl)pyridazine EGDMA Ethylene Glycol Dimethacrylate Et2O Diethylether EtAc Ethyl acetate EtOH Ethanol HEMA (2-Hydroxyethyl Methacrylate) MALDI-TOF Matrix Assisted Laser Desorption/Ionization Time Of Flight MeOH Methanol MeOD Methanol-d4 Mn Number Average Molecular Weight Mw Weight Average Molecular Weight NEt3 Trietylamine NMR Nuclear Magnetic Resonance PAOx Poly(2-alkyl/aryl-2-oxazoline) PEtOxn Poly(2-ethyl-2-oxazoline) with n repeating units PEtOxn-N3 Poly-α-methyl-ω-azido-(2-ethyl-2-oxazoline) with n repeating units PHEMA Poly(2-Hydroxyethyl Methacrylate) PMDETA N,N,N′,N′′,N′′-Pentamethyldiethylenetriamine PMeOx Poly(2-methyl-2-oxazoline) PMMA Polymethylmethacrylate SEC Size-Exclusion Chromatography SI Supporting Info Sym Symmetric TBAF Tetra-n-butylammonium fluoride TEG Triethyleneglycol THF Tetrahydrofuran TIPS Triisopropylsilyl
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1 Theoretical Part
1.1 Aim of the Thesis
Hydrogels are three-dimensional solid structures that contain a substantial amount of water.
1,2,3,4 The properties they have are interesting for different biomedical applications, including
tissue engineering and drug delivery.1,5 Self-healing hydrogels are hydrogels which have the
property to self-heal after the gel has been disrupted. Self-healing is a remarkable
phenomenon observed in nature. When tissue, skin or bone is damaged, it regains its former
structure. If a hydrogel has the ability to self-heal without external stimuli like in some
biological systems it will expand its potential application even broader. This makes the gel
injectable to the body, instead of direct implantations which is done with most non-self-
healing hydrogels. The aim of this project is to synthesize self-healing hydrogels with
supramolecular crosslinkers. The key element that makes all of these promising features
possible, is the type of crosslinker that will be used for this, namely, a supramolecular
crosslinker. A supramolecular crosslinker is a system for linking polymer chains together,
which relies on supramolecular interactions. In contrary to a covalent interaction, which binds
two atoms permanently, this non-covalent supramolecular bond is dynamic.6,7,8,9 There is an
equilibrium between the associated molecular assemblies and the individual molecules
resulting in continuous dynamic exchange between the supramolecular assembled entities
and non-assembled individual molecules. In this thesis, such a new supramolecular crosslinker
was developed. The entity that was used as supramolecular crosslinker is a supramolecular
metallogrid, where metal-ligand coordination is used as the dynamic bond. This type of
supramolecular interaction was chosen, because of the strong supramolecular bonds this
compounds forms, compared to the bonds of other supramolecular interactions. Another
advantage is that a grid structure, which has four ligands in one grid, gives the possibility of
crosslinking between different polymer chains.
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Figure 1: Left: Location of Fe(II) complexation by compound 8. Right: the pyridine analogue of compound 8 10.
The ligand designed for this study that comprises all desired aspects, compound 8, is shown
in Figure 1. Octahedral coordinating metals, including iron(II) and zinc(II), can be coordinated
by ligand 8 in water by the free electron pairs of nitrogen, on the locations that are shown in
Figure 1. These coordinated transition metal ions have three more binding sites free which are
available for binding with the second ligand to give full metal coordination. When the other
transition metal ion also does the same and all of this goes further on, a supramolecular grid
structure can be obtained as the grid structure represents a thermodynamic minimum with
maximal supramolecular interactions and still relatively high entropy (Figure 2). It should be
kept in mind that these grid structures are formed by supramolecular interactions, meaning
in this case, they are not always present like this, but they are in a dynamic equilibrium.
Sometimes the four iron atoms and four ligands have all coordination sites occupied,
sometimes they don’t. Analogues of compound 8 can be found in literature.10 These analogues
have two pyridines instead of the two triazole rings of our compound. With Fe(II)(BF4)2, these
analogues are known to yield supramolecular grid structures. The triazole ring in compound 8
is made by a copper catalyzed azide alkyne cycloaddition. Due to this reaction, different R
groups can easily be introduced, which is not the case for the already reported pyridine
analogues.
Figure 2: Schematic representation of the supramolecular grid structure formed by compound 8 with Fe(II) as
metal ion
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Within this thesis a stepwise approach will be followed to develop these self-healing hydrogels
as will be explained in the following manner, including the reasons why the different steps
were taken.
At first the synthesis of small molecules as R group, more specifically, triethyleneglycol will be
targeted to obtain water-soluble ligands. After the compound had been made,
supramolecular assembly of compound 8 in water will be tested with Fe(II) and Zn(II). Besides
oligomers, also polymers of different lengths will be attached to the ligand. The polymer
chosen for this task was poly(2-ethyl-2-oxazoline) (PEtOx), a polymer in which our group has
a lot of expertise, that is hydrophilic and in which it is straightforward to include an azide end-
group by endcapping of the polymerization. It will be tested if the polymeric ligands can still
form with Fe(II) and Zn(II) when polyethyloxazolines of different lengths are attached on it.
After completing all previous tasks, it will be possible to test the use of the grid structures for
making supramolecular hydrogels in water. Therefore the ligand will be reacted on one side
with a triethylene glycol oligomer and the remaining acetylene group will be attached to
polymers whit an azide functionality on both chain ends, instead of only one azide
functionality at one chain end. With this, a polymer having ligands at both chain ends will be
obtained to form a supramolecular network as shown in Figure 3. Finally, the hydrogel
properties and self-healing behavior should be evaluated.
Figure 3: Polymer chains interconnected by ligand 8 as crosslinker
The remainder of this theoretical part will introduce supramolecular interactions,
supramolecular metallogrids and self-healing hydrogels, together with applications of self-
healing hydrogels.
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1.2 Supramolecular Grid structures
The grid structures that are designed as crosslinker for hydrogels rely on supramolecular
interactions. This part will go more in details about these supramolecular interactions. In 1.2.1,
a brief introduction will be given to supramolecular interactions, in particular metal-ligand
interactions. Also the concept of self-assembly will be addressed here. 1.2.2 will talk more in
detail about the supramolecular metallogrids. 1.2.3 shows examples of how these grid forming
ligands behave in solutions of metal ions. Important aspects of compound 8 and its metallogrid
formation will be discussed.
1.2.1 Supramolecular Interactions by Metal-Ligand Interactions
Supramolecular chemistry is a different kind of chemistry compared to the covalent one.
Supramolecular forces are non-covalent physical interactions that act between different
molecules. These interactions can be directional and non-directional. They include host-guest
interactions, metal-ligand binding, hydrogen bonding, electrostatic interactions, π-π stacking
and hydrophobic effects. These supramolecular interactions all have different binding
strengths and strongly depend on the interaction partners as well as the conditions. The main
difference between covalent and non-covalent bonds is that these non-covalent interactions
are dynamic. The supramolecular bond is reversible, meaning that there is an equilibrium
between the molecules present as a supramolecular assembled entity and the molecules that
are not in this associated assembly.6,7,8 The metal-ligand interactions are the most important
concerning the formation of grid-structures.
Five parameters are important concerning supramolecular interactions. One describing the
equilibrium (the association constant Ka; equation (1) and (2)), two describing the binding
dynamics namely the rate of association (ka) and the rate of dissociation (kd) and the last two
being the concentration C and temperature T.11,12,13,14 Ka is a thermodynamic parameter which
describes the equilibrium between the molecules present as a supramolecular assembled
entity and the molecules that are not in this associated assembly. With this in mind, it can be
understood that a higher association constant represents a higher extent of formation of the
supramolecular entity. This constant is a widely used method for quantitating the affinity for
a supramolecular metal-ligand complex to be formed in solution. The Ka value of metal-ligand
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interactions are very high, although they strongly depend on the chosen metal and may range
from rather weak up to being stronger than covalent bonds.15 High supramolecular association
constants are in general difficult to achieve in aqueous solutions, which is the reason metal-
ligand interactions were chosen to act as a crosslinker for hydrogels as they are known to
remain strong in water.
The equation for Ka at a certain temperature is different for 1:1, 1:2, 2:1 etc. complexes. In a
1:1 complex, one metal ion forms a complex with one ligand and the same trend is set for 1:2
and 2:1 complexes.13 The equation for a 1:1 equilibrium, the most simple equilibrium (a), is
given in formula (1).13,16 The more general formula (2) is valid for all supramolecular metal-
ligand complexes in which m is the number of metal ions in one complex and n is the number
of the ligands in that one complex.13 Formula (1) is a special case of formula (2) in which m
and n are equal to one. As an example, the values m=1 and n=2 are filled in formula (2). A
complex consisting of more than two molecules isn’t likely to form in one step, but more in a
stepwise process. Formulas (3) and (4) are the association constants K1 and K2 for a 1:2
complex, with the equilibria (c) and (d) respectively.13 The more processes there are, the more
difficult to fit and get to the KA value.13,17
𝐾𝑎 =[𝑀𝐿𝑥+]
[𝑀𝑛𝑥+][𝐿]
(1)
𝑀𝑛𝑥+ + 𝐿 ⇋𝐾𝑎 𝑀𝐿𝑥+ + 𝑛(𝐻2𝑂) (a)
𝛽𝑚𝑛 =[𝑀𝑚𝐿𝑛]
[𝑀]𝑚[𝐿]𝑛 (2)
𝑚𝑀 + 𝑛𝐿 ⇋𝛽𝑚𝑛 𝑀𝑚𝐿𝑛 (b)
𝐾1 =[𝑀𝐿]
[𝑀][𝐿] (3)
𝐾2 =[𝑀𝐿2]
[𝐿][𝑀𝐿] (4)
𝑀 + 𝐿 ⇋𝐾1 𝑀𝐿 (c)
𝑀𝐿 + 𝐿 ⇋𝐾2 𝑀𝐿2 (d)
Four metals and four ligands participate in the formation of a [2x2] supramolecular grid. The
overall binding constant is found in formula (5).
𝛽44 =[𝐹𝑒4𝐿4𝑛
8𝑥+]
[𝐹𝑒2+]4[𝐿]4 (5)
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Experimentally, the Ka values at certain temperatures can be determined by measuring a
change in physical properties upon complexation, which can be done with methods like 1H
NMR spectroscopy, in which the chemical resonance is measured, and UV absorption, where
the absorption of the moieties is measured. ITC, isothermal titration calorimetry, is another
commonly used technique. ITC is, besides for measuring Ka, also commonly used for measuring
the stoichiometry of the formed supramolecular moieties. Other thermodynamic values,
including ΔG, ΔH and ΔS are also obtained with ITC.13,14,16,18
The degree of association at a given temperature,11 also called the degree of complexation,13
is dependent on both the value of Ka and on the concentration C of the supramolecular
moieties. This degree of association is proportional to [(KaC)1/2] 11. When talking about
hydrogels, the crosslink density in a supramolecular hydrogel can be estimated with this
equation.11
The supramolecular interactions of the [2x2] grid of our interest are dynamic, whereby the
individual moieties constantly associate to the supramolecular complex and constantly
dissociate back to their individual moieties. The rate at which this happens can be quantified
with ka and kd. The ratio of ka and kd leads back to Ka as shown in formula (6) and equation (e).
In a supramolecular hydrogel, a supramolecular crosslink will always fluctuate between an
active (associated supramolecular complex) and a dissociated crosslink (dissociated
supramolecular complex). 11,19 Different techniques exist to determine the kinetics of the
dynamic supramolecular exchange. The technique used depends on the time frame of the
dynamic process.6 For more information on how kinetic studies can be used to draw
mechanistic information, Bohn (2014)6 provides an interesting review.
𝐾𝑎 =𝑘𝑎
𝑘𝑑 (6)
𝑀 + 𝐿 ⇌𝑘𝑑𝑘𝑎 𝑀𝐿 (e)
Another important supramolecular concept that is important to discuss for these metal-ligand
interactions, is so-called self-assembly. Whitesides and Grzybowski defined self-assembly as
“the autonomous organization of components into patterns or structures without human
intervention.”20 The obtained supramolecular entity is formed spontaneously and depends on
external factors like the pH and temperature of the solution, the process is reversible and the
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assembly formed in the end is the thermodynamically most stable entity. A supramolecular
assembly can be kinetically inert and kinetically labile with as difference between these two is
that a labile coordination complex has an exchange of the ligands and metals in the solution,
whereby an inert coordination complex isn’t capable to do so. An inert coordination assembly
has a high activation energy for exchanging ligands. This inert coordination is kinetically
trapped and does not necessarily go to its most thermodynamically stable supramolecular
assembly.9 The grid structure of this thesis on the other hand, with coordination by Fe(II) or
Zn(II), is proposed to be kinetically labile and should be able to go to the thermodynamically
most stable compound, due to a relatively low activation energy. It should be noted that the
grid is not formed at once when the reagents are added, but a lot of intermediates are formed
before reaching the thermodynamic minimum of the supramolecular grid.9
In Figure 4, two examples of metal-mediated supramolecular self-assembled structures are
shown. An equilibrium between a supramolecular square and triangle (Figure 4; left) is
observed when adding Pd(NO3)2, diethylamine and the ligands shown in Figure 4 in solution.21
At higher concentrations, the amount of supramolecular triangles compared to squares
increases while decreasing the concentration increases the formation of supramolecular
squares compared to supramolecular triangles due to the terms of the thermodynamic
equilibrium. The square has a lower enthalpy compared to the triangle, while the triangle is
favored over the square when considering the entropy.22,21 Another example of a metal-
mediated supramolecular self-assembled structure is the supramolecular grid (Figure 5; right),
the structure of our interest. This self-assembled entity can be formed by the combination of
a transition metal and a ligand, for example Cu(I) with 3,6-di(2-pyridyl)pyridazine, also called
DPP (Figure 5; left), which is a widely used ligand for supramolecular grids.23
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Figure 4:Top: Equilibrium between a supramolecular square and triangle; Bottom: a supramolecular grid
structure. Reprinted from ref. 24
1.2.2 Supramolecular Metallogrids
Jean-Marie Lehn and Jack Harrowfield define that metallogrids ‘are oligonuclear metal ion
complexes in which the array of metal ions is essentially planar and each metal ion can be
considered to define a point in a square or rectangular structure.’25 To achieve such grid
structures, some design considerations need to be made. Besides the coordination number of
the metal, one also has to keep in mind the ligand’s coordination site. This coordination site
has to be perpendicular to the plane of the ligand at the metal centers.26 This coordination
setting at the site of the metal centers controls that a straight, stiff extension from one ligand
to a bigger ligand system will spontaneously give a two dimensional grid network, featured by
the metal ions lying in one plane.26 In practice, bidentate and tridentate subunits for the ligand
can be used in combination with tetrahedral, octahedral and sometimes bipyrimidal
coordinating metals. By imagining the structures, it can be understood that bidentate ligands
need a tetrahedral coordinating metal such as silver (I) or copper (I) and tridentate ligands
require an octahedral ion such as Fe(II), Zn(II) or Co(II) to get to the grid structure. In the case
of our grid forming compound, it is a tridentate ligand in combination with a octahedral ion.
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The ligands mostly contain nitrogen atoms, for their good donor qualities, but also examples
of oxygen and sulfur containing compounds exist. Bi- and terpyridines are used frequently as
basis for the grid-forming ligands. The ligands shown in Figure 5 can all chelate metals and
form metallogrids. During self-assembly, the increase in preorganization could give rise to
cooperativity.26 The intermediates formed during self-assembly are kinetically labile. At the
end, the thermodynamically most stable grid structure, together with the right metal ions, is
formed. On top of that, aromatic ring systems are rigid and capable of having π-π interactions,
which are an extra stabilizing force between the ligands when being in the grid structure. It
should be noticed that formation of a grid structure is thermodynamically favored, when
compared to other structures (for example polymer structures) albeit this will be
concentration dependent. A favorable enthalpy term comes from coordination of the metal
in the coordination site of the ligand (note that a non-cyclic polymer would have non-occupied
binding sites at the end). The grid structure is the structure which can be present with the
highest amount of discrete entities which have all coordination sites occupied, making it
entropically and enthalpically favorable. An important aspect to get to the desired grid
structure, is a necessity to force it to the right geometry, which is perpendicular, and a drive
to obtaining fully occupied binding sites. Moreover, internally there also has to be an imposed
orientation. Examples are steric effects, which suppress the formation of unwanted entities.
Another example of this is stabilizing interactions, for example π-π stacking, which orientate
the ligands to form the desired grid. Lastly, external factors like the presence of counterions
or solvent molecules also have to be kept in mind.26
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Figure 5: Ligands capable of forming grid structures by self-assembly 26,27,28,29
1.2.3 Supramolecular Grids in Solution
In solution, the supramolecular grid is not always that simple to form as some other entities
can also be present in solution. The grid formation is not unambiguous, being dependent on
the environment, the solvent, the concentration of the species, the counter anions of the
metal, the metal coordinating character and of course the identity of the ligand. In this part,
examples of ligands yielding grids and the limitations of some will be discussed.26
First of all, ligands designed for grid formation don’t necessarily form grids. The structure 2 in
Figure 5 can form a double helix, a triangle and a [2x2] grid.26 The reason for this is the poor
preorganization of the ligand. If the grid is the only structure that is wanted, a more
preorganized ligand is necessary. By changing the two inner pyridines by 4,7-phenantroline,
this preorganization can for example be introduced.
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Another parameter capable of altering the supramolecular entities is the counter anions of
the metal ions in solution. By templating anions inside the cavity of the formed supramolecular
structure, a grid can only be formed with the right anions. An example of this is ligand 1 in
Figure 5 (DPP), which was studied for its grid formation with non-matching tetrahedral
coordinating metal ions together with other anions present in solution. With Ni(II) or Zn(II) in
combination with BF4- or ClO4-, a [2x2] was formed. Having the bigger SbF6- in solution results
in the formation of, a pentagon (5 ligands together with 5 metals, as opposed to 4 ligands and
4 metals in the [2x2] grid). The reason for this observation is that the pentagon can fit the
bigger anion SbF6- inside the cavity instead of smaller ions like BF4- and ClO4-, which fit better
into the grid structure.27 This effect is called the templating effect.26
The combination of the valency of the metal and the coordinating character of the ligand has
also to be respected. Again studies on DPP, ligand 1 in Figure 5 were done. In combination
with Zn(II)perchlorate in acetonitrile, a tetrahedral coordinating ligand and a octahedral
metal, a triple helicate instead of a grid is obtained. It should be noted that this example is
unusual. The helicate motif normally forms with ligands containing flexible linkers whereas
the 3,6-di(2-pyridyl)pyridazine is a very rigid ligand.30
Another interesting observation is made on the large ligand shown in Figure 6, made by the
group of J.-M. Lehn. This ligand has coordination cavities which should make it possible to
form a [5x5] grid.31 Interestingly, the [5x5] grid was not observed in solution together with
Ag(I) in nitromethane. What is observed instead is a [4x5] grid (Figure 6). Baxter and Lehn give
three reasons for this behavior. First, the nitrogen atoms, when not forming a complex, are in
a so called transoid conformation, which is more stable than being in the cisoid conformation.
In a transoid conformation, the nitrogen responsible for coordination are trans to each other
over the C-C sigma bond which interconnects the pyridines. This transoid conformation is
more stable because the free electron pairs of the nitrogen atoms repel each other. For
forming a complex, they have to be in the less stable cisoid formation. Secondly, the N=N
bonds are shorter compared to C=C bonds, giving the ligands in the [5x5] grid a non-straight,
domelike shape, also disfavoring its occurrence. Lastly, the Ag(I)-N bond in the middle of the
[5x5] grid would have very long bond lengths. Combining all these factors make that the [4x5]
grid is formed. They also denoted it as a [2x(2x5)] grid because the ligands are placed on two
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sides, which can be more clearly seen in Figure 6.31 It should also be noted that the [4x5] grid
is not formed exclusively. A decanuclear helicate structure together with Ag(I) is also formed
indicating the formation of kinetically trapped structures.
Figure 6:Top: ligand designed to form a [5x5] supramolecular grid; Bottom: the formed [2x(2x5)]
supramolecular grid. Reprinted from ref. 31
The idea of using a Cu(I) catalyzed azide-alkyne cycloaddition between compounds and letting
the triazole-ring act as nitrogen-donor ligand instead of pyridine has been reported for
bipyridine32 and terpyridine33,34 analogues that could successfully be used for metal
coordination. Furthermore, there is one report of a triazole grid-forming DPP analogue. This
DPP analogue was prepared based on 3,6-(bisethenyl)pyridazine (figure 7) onto which an
oligoethyleneglycol was cycloadded yielding the grid-forming ligand. After the cycloaddition,
the obtained product successfully formed a [2x2] grid together with [Cu(CH3CN)4]PF6 in
dichloromethane (DCM). 35 In this work, we will use a related strategy to prepare terpyridine
like grid-forming ligands.
The examples given above all report the formation of grids in solvents other than water. In
this, we aim to make grids in water, this for opening the gate to biological applications. The
reason chosen for our ligand instead of DPP is because DPP doesn’t form grids in water with
Cu(I). This phenomena was discovered by studies on DPP with hydrophilic poly(ethylene
glycol) chains polymer groups on it (number 9, Figure 5). In DCM, this compound is able to
form the desired grid. In water on the other hand, the grid is not formed, even though having
hydrophilic polymer groups bound to the ligands. Reason for this behavior is a
disproportionation reaction, meaning, Cu(I) gets partially oxidized to Cu(II) and reduced to
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13
Cu(0). This result was apparent both when the Cu(I) was titrated to the ligand in a watery
solution and also when the grid was self-assembled in DCM first and subsequently transferred
to water. 29
Figure 7:Cu(I)AAC reaction for obtaining grid forming ligand
In summary, formation of only [nxn] grids without other supramolecular species being found
is negatively affected by having a ligand which can form complexes in the cisoid conformation.
The ability to form a domelike shape is also undesirable. But, like mentioned previously, a
drive to obtain the fully occupied binding sites by getting the maximum amount of
coordination, the highest amount of π-π stacking between ligands and the right choice of
anions, makes that exclusively forming supramolecular grids is possible.31 Ligand 8 is included
in Figure 5 as this compound is interesting for future work as hydrazides are relatively easy to
make.
1.3 Supramolecular Hydrogels
In polymer science, supramolecular chemistry and interactions is of major importance. The
special properties of polymers like Kevlar and nylon rely on cooperative non-covalent
interactions, more precisely on hydrogen bonding. For a lot of applications, including self-
healing hydrogels, the specificity and directionality of these non-covalent supramolecular
interactions can be used. Like mentioned before, this can be done by replacing covalent
crosslinks by directional dynamic non-covalent interactions. The use of supramolecular
chemistry and self-assembly in the polymer field is expected to be a promising path for smart,
adaptive and self-healing materials. Zhang et al. (2012)36 referred to supramolecular gels as
‘[…] the most promising materials in this field [..]’ when talking about self-healing hydrogels.
This also is the application what our supramolecular grid was designed for, to be used in
supramolecular self-healing hydrogels. In this part, hydrogels and in particular supramolecular
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hydrogels are introduced. Furthermore, the phenomenon of the shear-thinning effect and its
importance will be discussed.
1.3.1 Hydrogels
Hydrogels are three-dimensional structures that are held together by covalent or physical
crosslinks which can contain a substantial amount of water.1,2,3,4 The amount of water they
can absorb ranges from 10% to a thousand times their dry weight.37 The monomers for
building the polymer structures are hydrophilic, examples being carboxyl, amino and hydroxyl
groups. These hydrophilic groups are the main reason for the hydrogel being able to hold
water. Water holding capacity depends on the amount of hydrophilic groups as well as the
crosslink density. Higher crosslink density leads to a less stretchable polymer network and
therefore a lower maximum swelling capacity.38 Besides water, also nutrients, medicines and
other compounds of interest can be inserted in the hydrogel as aqueous solutions.1,5
Different classifications for hydrogels exist,1,38,11 the type of polymer chain can be used for
instance, namely ionic or neutral chains.11 More useful for our application is the type of
crosslinking, which can be permanent or non-permanent. In a permanent hydrogel, the
crosslinking is covalent. Because of this, these hydrogels are also called chemical hydrogels.
An example are PHEMA (poly(2-hydroxyethyl methacrylate)) hydrogels as used for contact
lenses. A crosslinked hydrogel of PHEMA is made by radical polymerization, for which HEMA
(2-hydroxyethyl methacrylate) is polymerized together with EGDMA (ethylene glycol
dimethacrylate) as bifunctional crosslinker.37 Other reactions for crosslinking include Michael
type additions, 1,3-dipolar cycloadditions of alkynes and azides and Schiff base formation.11
The equilibrium swelling in an aqueous solution of such covalent hydrogels is depended on
the crosslink density that can be quantified with MC, which is a parameter to approximate the
average molecular weight between the crosslinks of the hydrogel.37 Chemical crosslinking has
the advantage that optimization for the required application is easy. It is mostly used when
the application requires though and stable hydrogels.11 Limitations of permanent hydrogels
are the chemical techniques used for crosslinking leading for instance to traces of metal
catalysts, functional groups of unreacted crosslinker or photoinitiators. Also the use of UV light
for initiating the crosslinking has its disadvantage, for example in vivo crosslinking is something
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15
which is not possible.11 Other limitations are the permanent shape making them non-reusable
and non-reshapeable.
A non-permanent hydrogel is called a physical, dynamic, reversible or a supramolecular
hydrogel.1,37,38,7 The driving force for gel formation is molecular self-assembly.11 The forces
holding together the hydrogels are secondary forces, such as hydrogen bonding, ionic
interactions, and/or molecular entanglements,37,38 which all are transient forces.37,11 Gel
formation can be induced in water, without a pronounced change of volume. Consequently,
no crosslinking reagents are necessary. Instead of permanent crosslinking during the synthesis
of the chemical hydrogel materials the crosslinks are dynamic and they are constantly binding
and dissociating. The consequence of this is that a weaker gel is obtained, which can be more
easily sheared and reversibly deformed by mechanical forces. This dynamic character also has
its advantages as it gives self-healing and shear-thinning properties to the hydrogel.11,1
Examples are PVA-glycine hydrogels, gelatin and peptide hydrogels. Stimuli-responsive
hydrogels can change their equilibrium swelling by a change in surrounding environment.
Examples of these changes are pH and temperature.39,40,41
Figure 8: Comparison of a chemical (a.) and a dynamic hydrogel (b.). Reprinted from ref. 11
1.3.2 Self-Healing Hydrogels
Self-healing polymer gels use dynamic bond formation, meaning that a bond between
different polymer chains dissociates and recombines in a dynamic equilibrium. Most self-
healing hydrogels are physical hydrogels which mostly rely on non-covalent forces as ‘cross
linkers’. These secondary forces are non-covalent binding motifs, which can be specific and
directional.11 The supramolecular interactions used include host-guest, multivalent ionic,
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16
metal-ligand, hydrogen bonding, formation of stereo complexes and biomimetic interactions.
Different supramolecular interactions have different binding strengths and dynamics and one
can choose the right supramolecular interaction depending on the application of interest.
Inclusion complexes of cyclodextrins have moderate association constants (Ka) while metal-
ligand complexes have a high association constant, therefore our choice went to metal-ligand
interactions. With this in mind, it can be understood that a higher association constant
represents a stronger formation of the supramolecular entity. Cucurbit[n]urils are also
interesting for hydrogels because their binding strength can be varied over a range of 10
magnitudes depending on the cucurbit[n]urils and chosen guest.11
It is worth mentioning that not all self-healing gels are physical hydrogels. Chemical self-
healing gels have also been developed based on dynamic covalent bonds, for example
disulfide bonds or reversible Diels-Alder cycloadditions.42 Also, not all physical hydrogels rely
on dynamic secondary forces, an example being self-healing gels based on crystallization and
polymer-nanocomposite interactions resulting in kinetically trapped hydrogel structures.42
Figure 9: Different compounds with different Ka values. Reprinted from ref. 11
1.3.3 Applications of Self-Healing Hydrogels
Hydrogels have properties which are interesting for different biomedical applications,
including tissue engineering and drug delivery. In tissue engineering, the replacement of
organs by in vitro grown man-made organs, the hydrogel can be used as scaffold for cell
growth.43 For their use in drug delivery, drugs can be loaded into the pores and released at
the designed rate.44 An important requirement for implants is biocompatibility, to which the
high water content of hydrogels is beneficial. The drugs can be distributed to the specific tissue
of implantation but systematic delivery is also possible. It has to be kept in mind that also the
hydrogel forming material itself shouldn’t give physiochemical problems in the extracellular
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17
matrix, where it is implanted, and should have mechanical resemblances to the place of
implant.1,5
A method for introducing the hydrogel into the body is by direct implantation of the
crosslinked material in the body, in other words, the drug containing crosslinked hydrogel is
made in advance outside the body before being implanted. A more convenient method is to
inject the drug containing hydrogel through a needle, instead of implanting it by surgery. This
is where physically crosslinked hydrogels, like ours, could be used for because of having shear
thinning properties,1,5 which is a decrease in viscosity when undergoing an increase in shear
strain. Physically crosslinked hydrogels are often mechanically less strong compared to
chemically crosslinked hydrogels, but have shear-thinning and self-healing properties, make
them interesting for biomedical applications. Furthermore, soft injectable hydrogels are less
susceptible to damage than more tough hydrogels. The ease of tunable mechanical properties
and good flow and recovery make injectable hydrogels a good candidate for drug delivery
applications.5
Shear-thinning properties can be characterized via rheology measurements. With this
method, the effect of shear rate on viscosity and shear stress can be obtained. In Figure 10
left, an example of such an experiment is given. The hydrogel used in this example is a
supramolecular hydrogel consisting of a host polymer, CD-HA (cyclodextrin modified
hyaluronic acid), and a guest polymer, Ad-HA (adamantane modified hyaluronic acid). In the
graph, the closed symbols are the data of shear stresses and the open symbols represent the
viscosity, this for different weight percentages of Ad-HA in CD-HA. The evolution of the shear
stress and viscosity as a function of shear rate clearly indicates shear-thinning behavior by a
decrease in moduli with increasing shear rate.5 The shear rate that the hydrogel will undergo
when injected with a syringe is thought to be higher than tested here and will lead to a further
decrease in viscosity.5
After being injected, the hydrogel needs to recover to form the crosslinked material by self-
assembly. The timescale in which this happens is important, mostly a faster recovery is more
desirable to avoid loss of material by diffusion. Some hydrogels take several minutes or even
hours to recover from shear stress, which limits their applicability.5,45 The cargo in the
hydrogel and the hydrogel material itself can diffuse out of the material before it is recovered
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18
and therefore not using the method to its full capacity. Some complementary binding motifs
have very fast recoveries, including the Ad-HA and CD-HA host-guest system discussed above
which has ‘a near-immediate recovery following shear-thinning delivery, allowing optimal
retention of both material components and potential therapeutic cargo at the target site.’5 To
come to this conclusion, Rodell et al. exposed the material to cycles of oscillatory strain,
alternating a large amplitude and a low amplitude (Figure 10 right), in which the fast recovery
can be observed independent of the number of cycles.5
Figure 10: Left, Rheology measurement of Ad-HA and CD-HA; Right, oscillatory strain experiment, the full
black line is the applied oscillating strain, the red and blue lines are the G’ storage and G” loss modulus
respectively. Reprinted from ref. 5
1.4 Supramolecular Grids in Polymers and Gels
The supramolecular grids mentioned in 1.2 were not yet incorporated in polymer structures,
except the triazole DPP analogue. The question arises if these grid structures can also be
formed when they are part of a polymer chain. The DPP ligand A (Figure 11) immediately yields
a supramolecular grid with tetrakisacetonitrile Cu(I) hexafluorophosphate in DCM. Whether
ligands B, C and D (Figure 11) could also form a grid structure was investigated by UV-Vis
titration studies. The supramolecular complexation model, starting from metals and ligands in
solution, to the supramolecular grid structures, is rather complicated as a large number of
intermediate complexes can be formed. A simplified mechanism can be considered by
equilibria (f) and (g). In the case of B, the complex that is most present below 0,1
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19
stoichiometric equivalents of Cu(I) is L2Cu, whereas going to more than 0.1, the L4Cu4 grid was
most prevalent indicating strong cooperativity in the grid formation. In the case of C and D,
the shift to the grid being the most prevalent entity was only observed above 0.5 equivalences
of Cu(I). The proposed explanation given for the different behavior of C and D compared to B,
was that more sterical hindrance in C and D suppresses the cooperativity effect. Therefore,
the grid of B is formed at lower Cu(I) concentrations and is also stronger than grids of C and
D.17
2 𝐿 + 𝐶𝑢(𝐼) 𝑘1→ 𝐿2𝐶𝑢(𝐼) (f)
2 𝐿2𝐶𝑢(𝐼) + 2 𝐶𝑢(𝐼)𝑘2→ 𝐿4𝐶𝑢(𝐼)4 (g)
Figure 11: Ligand A, B, C and D
Another example makes use of ligand 8 (Figure 5), a bis(acylhydrazone), with Zn(II) as the d-
metal for coordination.28 This compound is capable of forming an organogel in toluene. The
MGC, minimum gelation concentration, or the lowest concentration of the gelator that is
needed to form a gel, was sought. In toluene, above ambient temperature, this was 18 mg/ml.
Heating of the gel above 38 °C makes the gel transform to a sol. By changing the concentration,
the gel to sol transition temperature could be changed. At a concentration of 25 to 35 mg/ml,
the transition temperature was found to be 44 °C. Higher concentrations were not tested. By
using 1H-NMR and IR, it could be determined that the grid structure was present in the
organogel. A DSC measurement on the sol-gel transition was also performed with a 20 mg/ml
concentration of ligand 8, to gain insight in the mechanism. Upon heating, an endothermic
peak was found between 35 to 67 °C. Cooling resulted in a exothermic peak between 48 and
18 °C, on which the authors state that this “demonstrates the full thermoversibility of the self-
assembly process.”28
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1.5 Supramolecular Metal-Ligand Interactions in Hydrogels
The idea of using grid like metal-ligand interactions as a supramolecular crosslinker in
hydrogels has not been explored, but related concepts exist. In early work, hydrogels were
made by bipyridyl-branched poly(2-methyl-2-oxazoline) together with Fe(II) and Ru(III) salts
by metal ligand complexation, when being at low temperatures and high concentrations.46 In
more recent work, terpyridine systems are frequently used. An example is a study of
terpyridines at the ends of an eight armed PEG star polymer yielding hydrogels with Fe(II) and
Ni(II).47
Another frequently used systems are catechols and comparable structures on star-polymers
together with Fe(III). These systems are also common when discussing self-healing. One major
advantage is their high association constant making them suitable for use in water under
ambient conditions and thus hydrogels. Tris- and bis- catechol-Fe(III) complexes have a
stability constant of Ks~1040. The amount of force needed to break this bond is only slightly
lower compared to breaking a covalent bond.15 With the information of these articles, it is
clear that making a self-healing gel with metal-ligand interactions as crosslinker, is possible.
To make these catechol based hydrogels, a four-armed star-polymer, PEG- DOPA4 with an
Mw~10000 is used together with FeCl3 with the ratio of FeCl3 DOPA being 3:1. DOPA stands for
dihydroxy-phenylalanine and is an amino acid which has a catechol as side group. An
advantage of this polymer is that the properties can be changed by altering the pH. At pH of
5, it is a green/blue fluid with mono-catechol complex while raising the pH to 8 leads to a
purple gel consisting of the di-catechol-Fe complex. At pH 12, a red gel of the tris-catechol-
complex is present. A change of color isn’t the only useful aspect, the properties of the gel
also change by pH. At pH 5, the system behaves viscous, so more like a fluid. The gels at pH 8
and pH 12 behave as elastic gels.15 The self-healing properties were also tested. To do so, a
covalent crosslinked gel was made by oxidizing PEG-DOPA4 with NaIO4. When comparing this
covalent gel to the supramolecular hydrogel, it turned out only the supramolecular hydrogel
intrinsic healed when they are damaged. The healing time was in the order of minutes.15
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One major problem concerning catechol-Fe(III) based hydrogels is the gel instability in time.
Catechol can oxidize to quinone and this can react with catechol, forming a covalent crosslink.
On an hour time scale, this is not a problem,15 but on a month time scale, it is.48 As a result,
the supramolecular self-healing hydrogel loses its excellent properties in time. To overcome
the disadvantage of instability in time, analogues for DOPA were searched and 3-hydroxy-4-
pyridinonone (HOPO) turned out to be a good one. When used on a PEG star-polymer with
the same molecular weight as used for DOPA and using Fe(III) as metal ion, no instability in
time was found.48
This PEG-HOPO4 also has a gelation at physiological pH, instead of the need for a high pH like
DOPA polymers, making it interesting for injecting in the body. To test this hypothesis, it was
injected in a buffer solution of physiological pH. It turned out that by doing this, a good gel
was obtained, opening its way to biological applications.48
1.6 Poly(2-oxazoline)s
Lastly poly(2-oxazoline)s will be introduced as this is the polymer chosen for cycloaddition to
obtain the polymeric grid forming ligand. Poly(2-oxazoline)s can be made by the ring opening
polymerization of 2-oxazolines. In these compounds the R group (Figure 12) can be altered
and chosen to obtain the desired properties.49,50 It can be simple alkyl chains like a methyl or
an ethyl which results after polymerization in hydrophilic polymer. n-Hexyl as R group on the
other hand yields a hydrophobic low glass transition temperature polymer.49 Besides simple
alkyls, also chemically more interesting moieties can be introduced on this place, including
functional handles like alkenes and esters.50
Polymerization of 2-oxazolines is done by cationic ring opening polymerization (CROP), which
is a living polymerization.49,50 No intrinsic termination occurs in contrast to controlled radical
polymerization. Also, the repellence of the growing cationic polymer chains suppress reaction
between the two growing polymer chains and hinder termination, making high monomer
conversion possible.50 Thermodynamically all polymerizations are unfavorable in terms of
entropy, but the polymerization of 2-oxazolines has a negative enthalpic term due
isomerization of the iminoether to a more stable amine functional group. In total, the
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22
enthalpic term is more pronounced and polymerization is thermodynamically favorable after
initiation.50
Figure 12: Polymerization mechanism of EtOx with IX as initiator and TY as terminator
Initiation of the polymerization is carried out by nucleophilic attack on the monomer (Figure
12). Lewis acids51, chloroformates52 and alkylating agents can be used as initiators. Nowadays,
alkylating agents like methyl tosylate are most popular.50 The identity of the counterion alters
the equilibrium of active cationic and non-active covalent entity and thereby the
polymerization rate (Figure 13).50
Figure 13: Equilibrium between cationic and covalent propagating species
After the monomer is started by initiation (Figure 12), the propagation step takes place. This
step is the heart of a polymerization, as the growth from a small molecule to a polymer takes
place. In this step, the equilibrium shown in Figure 13 is still existing. The propagation rate kp,
depends on the counterion, solvent, the type of monomer and temperature. kp follows
Arrhenius’ law given by formula 7. With respect to the parameters in these formulas, the
molecular weight of the polymer at a given reaction time can be calculated.
ln ([M]0
[M]t) = kp[I]0t (6)
kp = A e−
EaRT (7)
Termination of the living polymer chains by adding an end capping agent can occur on the two
and on the five place. Terminating agents like azides50,53,54, thiolates and sodium hydroxide
attack in the five position while water attacks on the two and the five positions (Figure 14)50.
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23
As water is an effective terminating agent, the CROP of 2-oxazolines has to be carried out
under very dry conditions.55
Figure 14:Termination can occur on the two and on the five position of the oxazolinium chain end
Another interesting feature of polymerizing 2-oxazolines is the ease of introducing chain-end
functionalities. By choosing a terminating and initiator agent with the functionality of choice,
end-group functionalities can be introduced.50 Propargyl p-toluenesulfonate as initiator is an
interesting compound for introducing an acetylene end-group56. Having two azide end-group
functionalities can also be carried out. This can be carried out by an azide functionalized
initiator57 in combination with quenching the reaction with an azide. Another method is using
diiodoalkyl initiation. Subsequently, sodium azide can be added to terminate and get a
polymer with azide functionalities on both ends54 (Figure 15).
Figure 15: Synthesizing PMeOx (poly(2-methyl-2-oxazoline)) with azide functionalities on both chain-ends.
PAOx (poly(2-alkyl/aryl-2oxazoline)) are not only interesting because the polymerization of 2-
oxazolines is living, the obtained polymers have many applications in life sciences. Interesting
is the stealth behavior, meaning the body does not recognize them as body foreign and, thus,
no or limited immune response occurs.58 This stealth behavior make PAOx ideal for drug and
gene delivery.50 PAOx are also ideal for making hydrogels. Covalent PAOx hydrogels have been
used for drug delivery and as cell culture scaffolds.59 By choosing the right monomers,
hydrophobicity and other properties of the biocompatible hydrogels can be altered. Surface
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24
modifications for making non-fouling surfaces are another biomedically focused application
of PAOx.50
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2 Results and Discussion
2.1 Synthesis Route
The synthesis route used for synthesizing compounds 3 and 4 (Figure 16) was inspired on
literature.60 First, 2,6-dibromopyridine 1 is asymmetrically substituted with triisopropylsilyl
(TIPS) protected acetylene 2 which is done via Sonogashira coupling, to give 2-bromo-6-
[(triisopropylsilyl)ethynyl]pyridine 3. Compound 3 is then reacted with n-BuLi, giving a
bromine-lithium exchange. Treating the intermediate with tributyltin chloride (Bu3SnCl) yields
the stannylated compound 4. The stannyl containing molecule 4 is coupled with compound 5
via a tetrakis catalyzed Stille coupling, yielding compound 6. This final reaction has not been
reported in literature before. Lastly, the deprotection of 6 is done with tetra-n-
butylammonium fluoride (TBAF). The synthesis of compound 3 to 7 is discussed in section 2.2
ligand synthesis.
Figure 16: Synthesis route for ligand 8
For the synthesis of the functional ligand 8, a copper(I)-catalyzed azide alkyne cycloaddition
will be used. With this reaction, different azide functionalized compounds can be attached to
protoligand 7, directly yielding the novel triazole containing grid-forming ligand 8. These azide
compounds can be oligomers, small molecules or polymers and can be used for incorporating
the grid in a polymer network, the final purpose of this project. As for the azide functionalized
compound groups, 2-(2-(2-methoxyethoxy)ethoxy)ethyl azide 13 was prepared as water-
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26
soluble oligomer and azide functionalized polyethyloxazolines were prepared with 20, 50 and
100 repeating units as water-soluble polymers. The synthesis of these oligomers and polymers
is discussed in section 2.3. polymer and oligomer synthesis.
2.2 Ligand Synthesis
2.2.1 Synthesis of 2-Bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3
Figure 17: Sonogashira coupling
The first reaction step involves the asymmetric substitution of only one bromine atom of 2,6-
dibromopyridine 1, by a Sonogashira coupling reaction (Figure 17) in tetrahydrofuran (THF) by
a TIPS protected acetylene group. PdCl2(PPh3)2 and Pd(PPh3)4, which is also called tetrakis,
were both tested as catalyst for this reaction. PdCl2(PPh3)2 60 is the catalyst used in literature
while tetrakis is also commonly used in such cross-coupling reactions. The reaction worked
well with both catalysts.
These catalysts are very oxygen sensitive. Therefore, the reaction mixture needs to be oxygen
free. Different methods for getting the mixtures oxygen free were tested. A first one was by
bubbling the solvent together with 2,6-dibromopyridine 1, the catalyst, CuI and triethylamine
(NEt3) with Ar. After bubbling, the (triisopropylsilyl)acetylene 2 was added dropwise. Another
method used for getting the mixture oxygen free was by using a schlenk line with a vacuum
and Ar line. The powders in the reaction vessel were brought under argon. Subsequently, the
solvent and NEt3 were added with a syringe. Then the (triisopropylsilyl)acetylene 2 was added
dropwise. Good results were obtained using both methods.
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27
In the article used for the synthesis of this compound 3,60 a three times molar excess of 2,6-
dibromopyridine 1 to(triisopropylsilyl)acetylene 2 was used to favor the mono substituted 2-
bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3 instead of the disubstituted 2,6-
[di(triisopropylsilyl)ethynyl]pyridine 3A (Figure 18). After finishing and purifying the reaction
products, 2,6-dibromopyridine 2 is still intact and can be used again in another reaction,
making the excess not wasteful. Another reason number 2 is used in excess is the price of
those chemicals.
Figure 18: The three major compounds in the crude reaction mixture
The characterization of the three main compounds in the reaction mixture was not straight
forward. With ESI-LCMS, 2-bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3 can be identified.
Compound 1 and 3A were not ideal for LCMS analysis. The 2,6-dibromopyridine was possibly
too polar to be found with ESI-LCMS and elutes to fast while 3A adhered to the column and
gave a very broad trail instead of a nice peak as it is too apolar. GCMS gave counts for all three
compounds (Figure 19). 2,6-dibromopyridine comes of first with a retention time of 11.7
minutes. The next compound is 3A, following at a retention time of 16.67 minutes. The
molecular ion of this compound is not visible because the m/z is only measured to 400 Da.
When measuring to 600 Da, the compounds fragment too much to be clearly identified.
Therefore, qualitative analysis of this peak could not be done, but by excluding other peaks
from being the disubstituted pyridine 3A, this peak at 16.67 could be assigned. The desired
compound 3 eluted last from the column into the mass spectrometer. All three compounds
show a characteristic mass spectrum of an aromatic compound, meaning that only the
substituents give major fragmentation while the pyridine is stable. On a silica TLC plate with
n-hexane and 15% of ethyl acetate (EtAc), the three peaks of the different compounds were
also visible. The spots were identified via larger scale preparative TLC in combination with
GCMS and 1H NMR spectroscopy. The lower spot is molecule 1, the middle spot 3 and on top
is compound 3A.
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28
Table 1: tr values of GCMS, LCMS and rf values of TLC
Compound tr GCMS tr LCMS rf TLC
2,6-dibromopyridine 1 11.7 - 0.50 2-bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3 17.3 0.53-6.28 0.90 2,6-[di(triisopropylsilyl)ethynyl]pyridine 3A 16.7 4.600 0.73
Figure 19: GCMS results
In the 1H NMR spectra (Figure 20), the 2,6-dibromopyridine 1 peaks overlap with those of
compound 3. The conversion of the aromatic peaks is shown when purifying the crude mixture
B to the purified compound 3. Spectrum B is a superposition of 2 molar equivalences of 2,6-
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29
dibromopyridine 1 and 2-bromo-6[(triisopropylsilyl)ethynyl]pyridine 3. Upon integration, a
pure 1H NMR spectrum of 2-bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3 should integrate for
3 hydrogens in the aromatic area while the TIPS protecting group, consisting of three isopropyl
groups having a proton chemical shift of 1,12 and 1,13, should integrate for 21. By combing
1H NMR spectroscopy, TLC and LCMS analysis, unambiguous identification of the desired
molecule 3 can be done.
Figure 20:1H NMR spectra of compound 1 (A), the crude reaction mixture (B) and compound 3 (C). In the box, a
zoom of the aromatic area is shown.
In literature, the purification of 3 was described by column chromatography on silica with n-
pentane and DCM as eluent, starting from 90% n-pentane to 70% in the end. When using these
solvents, no clean product was obtained. The fraction containing most of the desired
compound 3 still had a reasonable amount of 2,6-dibromopyridine 1 in it, which is undesirable
for the next reaction step. On TLC with silica as stationary phase, these compounds came very
close to each other. To improve the separation other eluents were tested on TLC (Table 2).
These solvents were also tested on TLC’s with neutral aluminum oxide as stationary phase but
this resulted in bad retention and almost no separation. The best result was obtained with
silica using EtAc in n-hexane. n-Hexane with EtAc starting from zero percent of EtAc was
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30
chosen as eluent system for the column chromatography. The silica column was first run with
0.5% of NEt3 in n-hexane before loading the compounds, to make the silica less acidic. Even
with these optimized solvents, still no completely pure compound could be obtained. The
most pure fraction of compound 3 still had 1 and 3A as minor impurities in it (analyzed by
GCMS and TLC). In the 1H NMR spectrum, the aromatic area integrated for 3,93 hydrogens
when setting the integration of the TIPS groups to 21. Because there are 3 compounds
present, the disubstituted 3A and 2,6-dibromopyridine 1 being the minor ones and the desired
2-bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3 being the major one, it’s not possible to
unambiguously determine the percentage of the 2,6-dibromopyrimidine 1 that still is present
in the 2-bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3.
Table 2: TLC results of the crude reaction mixture with different eluents.
Eluents % second solvent rfA rfB rfC remark
n-pentane - - - - - one trail
n-pentane DCM 10 0,47 0,26 - only 2 spots
n-pentane DCM 20 0,83 0,58 0,46 spots
n-hexane - - 0,13 - - one spot
n-hexane CHCl3 10 0,42 0,38 - trail, only 2 spots
n-hexane DCM 10 0,21 0,14 - trail, only 2 spots
n-hexane DCM 20 0,49 0,38 0,08 trail
i-octane DCM 20 0,22 0,18 - peak overlap
n-hexane EtAc 4 0,81 0,59 0,39 spots
n-hexane EtAc 10 0,86 0,57 0,47 spots
n-hexane EtAc 15 0,90 0,73 0,50 spots
n-hexane EtAc 40 0,98 0,93 0,80 spots
As is already stated above, the disubstituted compound 3A does not give problems in the next
reaction step. Therefore, another less time consuming method, compared to column
chromatography, was searched to purify the crude mixture. The diiodopyrimidine 1 has got a
reasonable boiling point at standard pressure, being 255°C.61 This opens the way for other
purification methods, including vacuum distillations. A Kugelrohr setup, which is de facto a
short distance vacuum distillation with a rotating oven was used for removing 1 from the
crude. The pressures needed to evaporate the 2,6-diiodopyrimidine 1 out were calculated (SI
2). Pressures of 0.4 mbar could be reached with the used setup, making a temperature of 85°C
ideal to remove 2,6-dibromopyridine 1 from the crude mixture. Subsequently, it was also tried
to distil over molecules 3 and 3A. The high temperatures needed led to boiling of all content
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in the flask, by which also drops containing catalyst came over in the trap. Another problem
when distilling 3 and 3A over was that at temperatures above 150°C, a black tar was formed,
as it indicates degradation of the products. For the final purification method, the Kugelrohr
was used in a first step to distill all of the 2,6-dibromopyridine 1 out of the crude mixture.
Subsequently, a flash column chromatography was done on silica with 30% EtAc in n-hexane
to get rid of the catalyst and all the other entities present in the crude mixture. Following this
procedure, 2-bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3 was obtained, which only
contained a minor amount of 2,6-[di(triisopropylsilyl)ethynyl]pyridine 3A. In the 1H NMR
spectrum, the aromatic area integrates for 2,96 while the TIPS groups integrate for 21 (SI 5).
With this it can be calculated that the amount of 3A in 3 is around 3%. Even though this value
is not very reliable as the integrating on 1H NMR spectra has some inaccuracy, it does give an
idea of the amount of disubstituted pyridine 3A in our otherwise clean compound 3 that was
utilized for the next reaction.
2.2.2 Synthesis of 2-[(Triisopropylsilyl)ethynyl]-6-(tributylstannyl)pyridine 4
Figure 21: Substitution of Br with tributylstannyl by first a halogen-lithium exchange
followed by reaction with Bu3SnCl
2-Bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3 was first treated with n-BuLi to give a lithium
intermediate. Subsequently, tributyltin chloride (Bu3SnCl) was added to prepare the
stannylated compound 4. The conditions initially used for this reaction were based on
literature.60 A mixture of 3 in THF was treated with n-BuLi at -78°C after which it was allowed
to heat till -20°C. Bu3SnCl was added when the mixture was at -78°C again after which it was
allowed to heat to room temperature. In the first reaction attempt, the right product was not
obtained. The 1H NMR peaks are published in literature and contain a double doublet at 7,40
and two doublets at 7,28 and one at 7,27. These reported values did not correspond to those
of our reaction product (Figure 22).
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Figure 22: Crude 1H NMR spectrum of stannylation with literature conditions
The proposed compound that was formed is 2-[(triisopropylsilyl)ethynyl]pyridine 9 (Figure
22). With LCMS, the [M+H]+ was found at m/z 260 and also 1H NMR spectroscopy confirmed
the formation of 9 (Figure 22) as the proton chemical shifts of 2-
[(triisopropylsilyl)ethynyl]pyridine 9 correspond to those reported in literature.62 Initially,
impurities of compound 3 were blamed for failure of the reaction. However, further
purification of product 3 did not help. For a new reaction, a new dry bottle of Bu3SnCl was
used because there were questions about the purity of the old SnBu3Cl (SI 13) and the
solutions were prepared in the glove box. The same reaction conditions were used and the
obtained product was again compound 9.
To investigate what exactly went wrong with the reaction, it was tried to substitute the
bromine atom by something else instead of Bu3SnCl. A deuterium atom was used as analysis
of a deuterium substitution is easily seen by 1H NMR spectroscopy and LCMS. As such, it could
be checked if it was possible to do a simple substitution reaction on the bromine atom of 2-
bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3 after lithiation. For this deuteration reaction,
the same procedure was used as before yielding 2-[(triisopropylsilyl)ethynyl]-6-
(deuterio)pyridine 10 (Figure 23). This structure was confirmed with 1H NMR spectroscopy
(Figure 23) and with LCMS that revealed the [M+H]+ at m/z 261. Based on this model reaction,
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it could thus be concluded that the bromine/lithium exchange by n-BuLi is not the problem,
but the attack of the lithium intermediate on Bu3Sncl is.
Figure 23: Crude 1H NMR spectrum of deuterated product 10
The reaction of methanol-d4 (MeOD) with the lithium salt intermediate of compound 3 is
thought to be fast, giving rise to such a high amount of compound 10. In the stannylation
reaction, a hydrogen comes into the reaction mixture after successfully having added the n-
BuLi. A possible hydrogen source could be the degradation of THF. THF can namely be
deprotonated by strong organolithium bases.63,64,65
Other reaction conditions were searched to avoid this side reaction. In more simple
stannylation reactions on 2-bromopyridine with n-BuLi and tributyltinchloride, the reaction
has been reported to proceed at -78°C instead of ambient temperature.66,67 Furthermore, the
reaction mixture is often kept at-78°C before the tributyltinchloride is added, instead of first
allowing it to heat till -20°C. Using this optimized procedure, the desired 2-bromo-6-
[(triisopropylsilyl)ethynyl]pyridine 4 could successfully be obtained, which was confirmed by
1H NMR spectroscopy, 13C APT NMR spectroscopy and LCMS. Two major products are formed
using these new reaction conditions, namely compound 4 and 9. In Figure 21, the crude 13C
APT NMR is shown, the proton chemical shifts of both compounds 4 and 9 were also confirmed
(Figures SI 3 and SI 4). These 13C chemical shifts were reported in literature and were also
calculated and confirmed with ChemBioDraw 13 for redundancy.
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Figure 24: 13C APT NMR spectrum of Crude reaction mixture with new conditions
Purification of the stannylated compound 4 was done with column chromatography on
aluminum oxide with n-pentane as eluent. The 2-[(triisopropylsilyl)ethynyl]-6-
(tributylstannyl)pyridine was obtained with a yield of 26 %. The reported yield is 45 %.60
2.2.3 Stille Coupling Reactions
Figure 25 : Synthesis of 4,6-bis[4’-((triisopropylsilyl)ethynyl) pyrid-2’-yl]pyrimidine 6
For the synthesis of 4,6-bis[4’-((triisopropylsilyl)ethynyl)pyrid-2’-yl]pyrimidine 6, 2-[(tri-
isopropylsilyl)ethynyl]-6-(tributylstannyl)pyridine 4 should be coupled to 4,6-diiodopyrimidine
5. This can be done via a Stille coupling with the addition of a catalyst. This exact reaction is
not reported in literature. In literature, compound 4 is used together with 3,8-dibromo-4,7-
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phenanthroline in a Stille coupling with toluene as a solvent.60 4-Iodopyrimidine has also been
used in a Stille coupling with 3-bromopyridine in toluene under reflux.68 Tetrakis was used as
Pd catalyst in both instances. The coupling of compound 4 and 6 was attempted in toluene
under reflux and tetrakis as catalyst as similar reactions had been carried out under those
conditions. The mixture was stirred overnight and a black precipitation was formed. Analysis
was done by 1H NMR spectroscopy and LCMS. With LCMS, a very broad band on the baseline
was visible. Because compound 6 is very apolar, it could be that this compound sticks to the
column, making LCMS hard for the identification. To be completely sure that the desired
compound was not present in the reaction mixture, a column on the crude mixture was
performed. The fractions were analyzed via 1H NMR spectroscopy confirming that compound
6 was not formed.
Stille coupling reactions sometimes need reaction times of several days 68,69,70,71. The
possibility of a too short reaction time arose, because a lot of the stannylated compound 4
was still present in the crude reaction mixture. Therefore a new reaction was done with
toluene under reflux, but now it was allowed to react for eight days instead of one. While the
reaction was followed by 1H NMR, it was clear that during the reaction, some peaks changed
in intensity relative to each other. In Figure 23 the change of the relevant peaks is shown. As
can be seen, after 48 hours, no peak was present at 9.2, while after 96 hours a peak arose at
that particular shift. In the sample after 140 hours, this peak was even bigger. After eight days,
the reaction was stopped. Again, the reaction mixture was black and had a black precipitate.
After evaporating the solvent, column chromatography was performed on silica which was
first treated with NEt3. The eluent was DCM with methanol (MeOH) going from 0 till 10% of
MeOH with NEt3 added in the end. The different fractions were analyzed by 1H NMR
spectroscopy and the fraction that looked most promising was also controlled by HSQC but
the desired product could not be identified indicating that compound 6 could not be obtained
using these conditions. Unreacted 2-[(triisopropylsilyl)ethynyl]-6-(tributylstannyl)pyridine 4
was obtained and besides compound 4, also a lot of 2-[(triisopropylsilyl)ethynyl]pyridine 9 was
formed. This compound 9 is a thermal degradation product of 2-[(triisopropylsilyl)ethynyl]-6-
(tributylstannyl)pyridine 4, which due to the long reaction times can be formed when not
being able to couple to another species present in the reaction mixture.
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Figure 26: The 1H NMR spectra after 48, 96 and 140 hours. The peak at 9.32 changes relative to the peak
at 9,40.
Compound 5, which was not stored in a fridge, did not look completely clean in the 1H NMR
spectrum (SI 1). Also the LCMS analysis looked rather suspicious, as a lot of impurities were
found in what should have been a clean compound (SI 11 and SI 12). With HSQC, it was
determined that it nevertheless was mainly the right product. Therefore, the Stille coupling
was attempted with another compound, namely 4,6-dichloro-2-phenylpyrimidine 5A, also
called fenclorim, which has two chlorine atoms instead of the iodine atoms of 4,6-
diiodopyrimidine 5 (Figure 27). Fenclorim also has a phenyl ring instead of a hydrogen atom
on the carbon between the two nitrogen atoms. In prospect of the grid forming ability when
using compound 6A instead of 6, this phenyl group is rather interesting. Like the terpyridine
analogue of ligand 8, there also exists a terpyridine analogue with a phenyl group on that place
instead of a hydrogen. In the corresponding [2x2] grid structure, this phenyl group provides
π-π interactions that stabilize the supramolecular grid. Because this fenclorim 5A was stored
in a fridge and was more clean, together with the added value of having a phenyl group in the
middle, the next Stille coupling was carried out with fenclorim 5A instead of 4,6-
diiodopyrimidine 5.
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Instead of toluene, DMF was used, which is a more standard solvent for carrying out Stille
couplings compared to toluene72,73,74. Tetrakis and PdCl2(PPh3)2 were both used as Pd catalyst
and the new reaction scheme is displayed in Figure 27.
Figure 27: Synthesis of 4,6-bis[4’-((triisopropylsilyl)ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 6A
ESI-MS analysis of the crude pointed out that 4,6-bis[4’-((triisopropylsilyl)ethynyl)pyrid-2’-yl]-
2-phenylpyrimidine 6A was present in both the crudes at m/z 671,4. Because the spectra
looked more or less the same and both reactions were done on small scale, workup was done
on the combined product. First, a column on silica with n-pentane/DCM (DCM in a gradient
from 0 to 20 percent with a couple drops of NEt3 was carried out. The fraction containing
compound 6A was further purified by recrystallization with ethanol (EtOH) as it is a non-
solvent at room temperature and a solvent under reflux. 4,6-bis[4’-
((triisopropylsilyl)ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 6A was obtained with a yield of 57%
(Figure 28).
The Stille coupling with 4,6-diiodopyrimidine 5 was not carried out in DMF due to time
constraint. Further research is needed to find if the conditions for synthesizing compound 6A
also work with chemical 4,6-diiodopyrimidine 5.
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Figure 28: 1H NMR spectrum of 4,6-bis[4’-((triisopropylsilyl)ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 6A
2.2.4 Synthesis of 4,6-Bis[4’-(ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 7A
Figure 29: Deprotection of 6A to get to the protoligand 4,6-bis[4’-(ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 7A
The deprotection of 4,6-bis[4’-((triisopropylsilyl)ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 6A
was done with TBAF. In literature, 1.3 to 2.5 equivalents of TBAF to one TIPS acetylene group
are used.60,75 Here, 1.8 equivalents of TBAF for one TIPS group was used and the reaction was
followed by TLC. After one hour, the mixture didn’t change anymore and after one and a half
hour, the reaction was stopped. Recrystallization was tried in EtOH since compound 7A was
suspected to be a crystalline compound. No crystallization occurred so another method was
searched. Washing the crude with water, diethylether (Et2O) and n-pentane was another
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literature inspired 60 workup which also did not prove to be successful. Likewise a simple
extraction with water and chloroform was non satisfying as well. Lastly, a column on silica with
n-pentane/DCM/MeOH in a gradient starting from 85/15/0 to 0/90/10 with 0.5 % of NEt3 was
performed. After evaporation, protonated NEt3 was in it, which was removed by resolving the
product in CHCl3 and washing with water. The right structure was proved by 1H NMR
spectroscopy (Figure 30) and confirmed by ESI-MS that revealed the [M+H]+ at m/z 359.2. The
protoligand 7A was obtained with a yield of 97%. It still contained impurities in the lower ppm
area.
Figure 30: 1H NMR spectrum of 4,6-bis[4’-(ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 7A
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2.3 Polymer and Oligomer Synthesis
The aim of synthesizing these oligomers and polymers is to attach them onto alkyne
functionalized ligand 7 via a cycloaddition. The polymers and oligomers should have an azide
functionality as end-group. Synthesis of 2-(2-(2-methoxyethoxy)ethoxy)ethyl azide 13 is
discussed in section 2.3.1 and the poly-α-methyl-ω-azido-(2-ethyl-2-oxazoline) (PEtOx-N3)
synthesis in section 2.3.2.
2.3.1 Synthesis of 2-(2-(2-Methoxyethoxy)ethoxy)ethyl Azide 13
Figure 31: Synthesis of azide functionalized monomethyl triethylene glycol
2-(2-(2-Methoxyethoxy)ethoxy)ethyl azide 13 was synthesized starting from triethylene glycol
monomethyl ether (TEG) 11 (Figure 31). Firstly, the TEG 11 was tosylated by TsCl. The used
conditions came from literature76 and the reaction was carried out in DCM. NEt3 was added in
excess and 4-dimethylaminopyridine (DMAP) was added substoichiometric as catalyst. After
workup, which involved extraction with water, purification was performed by column
chromatography. The tosylated monomethyl triethylene glycol 12 was obtained with a yield
of 70%. Analysis was done by 1H NMR spectroscopy and LCMS. The reported 1H NMR peaks
were visible and no impurities were found (SI 9). With LCMS the [M+NH4]+ peak was observed
instead of the [M+H]+ peak. Small ethylene glycol chains can act as supramolecular podands,
making them able to coordinate cations,9 such as ammonium cations. In an environment with
such a high concentration of ammonium compared to the concentration of compound 12 as
ammonium is present in the eluent, observing only the [M+NH4]+ peak is accountable.
Upon tosylation of the triethylene glycol monomethyl ether 12, the primary alcohol group of
compound 11 was transferred in a better leaving group. In the next step, a nucleophilic
substitution reaction was carried out. This reaction was also reported in literature before and
was performed with 3.5 equivalents of NaN3 in DMF at 60°C.77 After extraction with Et2O,
triethylene glycol monomethyl ether azide 13 was obtained with a yield of 84%. The expected
1H NMR shifts were found (SI 9) and the compound was confirmed with ESI-MS with the
[M+H]+ at m/z 190.2 Da and the [M+NH4]+ at 207.2 Da.
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2.3.2 Synthesis of Poly-α-methyl-ω-azido-(2-ethyl-2-oxazoline)
Figure 32: Synthesis of PEtOx-N3 by CROP and termination by sodium azide
PEtOx-N3 was made by polymerization of 2-ethyl-2-oxazoline with MeOTs as initiator. As the
CROP of 2-oxazolines is a living polymerization that is very sensitive to nucleophiles it requires
working under dry conditions for which the reaction was carried out in a glovebox. After
polymerization, sodium azide is added as terminating agent to introduce the azide end-
group.50,53,54 Three different lengths of PEtOx-N3 were made, namely with 20, 50 and 100
repeating units. The standard optimized conditions of our group were used, being 0.05
equivalents of MeOTs and 0.15 equivalents of NaN3 with regard to 2-ethyl-2-oxazoline in ACN
at 100°C for PEtOx20-N3. For PEtOx50-N3 these equivalences where 0.02 MeOTs and 0.06 NaN3,
for PEtOx100-N3 0.005 MeOTs and 0.03 NaN3 for. After polymerization, the two times molar
excess of NaN3 had to be removed from the crude. First, an extraction with EtAc and water
was used as workup. The resulting solid was dissolved in EtAc and extracted with saturated
NaHCO3 and saturated NaCl solutions for two times. The amount of the PEtOx-