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
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
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.
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
2
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
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
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
8
Supporting Info ................................................................................................................. 89
Abbreviations
Asym
Bu3SnCl
CuAAC
DCM
DMA
DMAP
DPP
EGDMA
Et2O
EtAc
EtOH
HEMA
MALDI-TOF
MeOH
MeOD
Mn
Mw
NEt3
NMR
PAOx
PEtOxn
PEtOxn-N3
PHEMA
PMDETA
PMeOx
PMMA
SEC
SI
Sym
TBAF
TEG
THF
TIPS
Asymmetric
Tributyltin chloride
Cu(I)-Catalyzed Azide-Alkyne Cycloaddition
Dichloromethane
N,N-Dimethylacetamide
4-Dimethylaminopyridine
3,6-Di(2-pyridyl)pyridazine
Ethylene Glycol Dimethacrylate
Diethylether
Ethyl acetate
Ethanol
(2-Hydroxyethyl Methacrylate)
Matrix Assisted Laser Desorption/Ionization Time Of Flight
Methanol
Methanol-d4
Number Average Molecular Weight
Weight Average Molecular Weight
Trietylamine
Nuclear Magnetic Resonance
Poly(2-alkyl/aryl-2-oxazoline)
Poly(2-ethyl-2-oxazoline) with n repeating units
Poly-α-methyl-ω-azido-(2-ethyl-2-oxazoline) with n repeating units
Poly(2-Hydroxyethyl Methacrylate)
N,N,N′,N′′,N′′-Pentamethyldiethylenetriamine
Poly(2-methyl-2-oxazoline)
Polymethylmethacrylate
Size-Exclusion Chromatography
Supporting Info
Symmetric
Tetra-n-butylammonium fluoride
Triethyleneglycol
Tetrahydrofuran
Triisopropylsilyl
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-selfhealing 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.
1
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
2
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 endgroup 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 selfhealing hydrogels.
3
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
4
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 metalligand 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 metalligand 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
[𝑀𝐿𝑥+ ]
𝐾𝑎 = [𝑀𝑥+ ][𝐿]
𝑛
𝑀𝑛𝑥+ + 𝐿 ⇋𝐾𝑎 𝑀𝐿𝑥+ + 𝑛(𝐻2 𝑂)
[𝑀 𝐿 ]
𝑛
𝛽𝑚𝑛 = [𝑀]𝑚
𝑚 [𝐿]𝑛
𝑚𝑀 + 𝑛𝐿 ⇋𝛽𝑚𝑛 𝑀𝑚 𝐿𝑛
[𝑀𝐿]
𝐾1 = [𝑀][𝐿]
[𝑀𝐿 ]
2
𝐾2 = [𝐿][𝑀𝐿]
(1)
(a)
(2)
(b)
(3)
(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).
[𝐹𝑒 𝐿 8𝑥+ ]
4 4𝑛
𝛽44 = [𝐹𝑒 2+
]4 [𝐿]4
5
(5)
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.
𝑘
𝐾𝑎 = 𝑘𝑎
(6)
𝑑
𝑘
𝑀 + 𝐿 ⇌𝑘𝑎𝑑 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
6
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 metalmediated 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
7
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.
8
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
9
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.
10
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
11
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
12
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 selfhealing 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
13
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
14
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 selfhealing 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,
15
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 metalligand 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 selfhealing 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
16
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 selfassembly. 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
17
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
18
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
𝑘1
2 𝐿 + 𝐶𝑢(𝐼) → 𝐿2 𝐶𝑢(𝐼)
𝑘2
2 𝐿2 𝐶𝑢(𝐼) + 2 𝐶𝑢(𝐼) → 𝐿4 𝐶𝑢(𝐼)4
(f)
(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 dmetal 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 selfassembly process.”28
19
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-catecholcomplex 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
20
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-4pyridinonone (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
21
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. k p 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.
[M]
ln ( [M]0 ) = k p [I]0 t
(6)
t
Ea
k p = A e−RT
(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.
22
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 2oxazolines 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
23
modifications for making non-fouling surfaces are another biomedically focused application
of PAOx.50
24
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-nbutylammonium 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 water25
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 2,6dibromopyridine 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.
26
In the article used for the synthesis of this compound 3,60 a three times molar excess of 2,6dibromopyridine 1 to(triisopropylsilyl)acetylene 2 was used to favor the mono substituted 2bromo-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.
27
Table 1: tr values of GCMS, LCMS and rf values of TLC
Compound
2,6-dibromopyridine 1
2-bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3
2,6-[di(triisopropylsilyl)ethynyl]pyridine 3A
tr GCMS
11.7
17.3
16.7
tr LCMS
0.53-6.28
4.600
rf TLC
0.50
0.90
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 2,628
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 npentane 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
29
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
n-pentane
n-pentane DCM
10
n-pentane DCM
20
n-hexane
n-hexane
CHCl3
10
n-hexane
DCM
10
n-hexane
DCM
20
i-octane
DCM
20
n-hexane
EtAc
4
n-hexane
EtAc
10
n-hexane
EtAc
15
n-hexane
EtAc
40
rf A
0,47
0,83
0,13
0,42
0,21
0,49
0,22
0,81
0,86
0,90
0,98
rfB
0,26
0,58
0,38
0,14
0,38
0,18
0,59
0,57
0,73
0,93
rfC
0,46
0,08
0,39
0,47
0,50
0,80
remark
one trail
only 2 spots
spots
one spot
trail, only 2 spots
trail, only 2 spots
trail
peak overlap
spots
spots
spots
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
30
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).
31
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 SnBu 3Cl (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 2bromo-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,
32
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 nBuLi. 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.
33
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-[(triisopropylsilyl)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-4,734
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.
35
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 4,6diiodopyrimidine 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 4,6diiodopyrimidine 5.
36
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 nonsolvent
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.
37
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
38
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
39
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.
40
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 endgroup.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-N3 going to the
EtAc phase was relatively low, as PEtOx is a hydrophilic polymer. The EtAc fraction after
evaporation had a white, non-transparent look, pointing to a non-salt free polymer. As this
workup was not satisfying, the watery NaHCO3/NaCl and EtAc fraction, were combined and
purified by a PD-10 desalting column to get rid of the NaN3 and the NaHCO3 and NaCl that
were in the water phase with which the extraction was performed. This PD-10 is de facto a
preparative size exclusion chromatographic column, made for desalting biomolecules. The
standard procedure could be used for desalting the polymers with 100 repeating units. For
the PEtOx50 and PEtOx20, a non-standard procedure was needed for desalting the aqueous
solution (more info about the procedure is given in experimental) to avoid losing the product
in the waste fractions. After desalting, the polymer was freeze dried and 1H NMR spectroscopy
and DMA-SEC (size-exclusion chromatography with N,N-dimethylacetamide as solvent)
analysis were carried out. The 1H NMR spectrum was first taken in DMSO-d6 in which one of
41
the chemical shifts of DMSO overlapped with the chemical shift of the backbone –N(C=O)CHa2-CHa2 hydrogens (SI 6). In the CDCl3 spectrum, a peak around 2.15 was visible, which
overlapped to some extent with the (C=O)-CHb2-CHc3 hydrogens. As this shift is rather high for
coordinated water in chloroform-d, a new spectrum on the same sample was taken with a
drop of water added. The sample went to a two phase system and the peak at 2.15 shifted (SI
7) while a peak at 4.75 arose, which is the normal chemical proton shift for pure water,
indicating that the peak at 2.15 was the water peak.
Figure 34: 1H NMR spectrum of PEtOx50-N3 measured in CDCl3
The hydrogens of carbon d are from the initiating group and by comparing the integration of
hydrogens b and hydrogens d, the number average molecular weight (Mn) was calculated
(Table 3 and formula 8). The 1H NMR spectra of PEtOx20-N3 and PEtOx100-N3 are added in SI 8.
Mn
proton NMR
= MCH3 + n Mrepeating unit + MN3
= 15 + (
3
∫ protons b
∗
) 99 + 42
4
∫ protons d
Formula 8: Calculation of Mn via 1H NMR spectroscopy.
42
PEtOx20-N3
11000
PEtOx50-N3
10000
PEtOx100-N3
9000
RI Response
8000
7000
6000
5000
4000
3000
2000
1000
0
25
26
27
28
29
30
31
32
33
34
35
Retention time (min)
Figure 35: DMA SEC data of PEtOx20 PEtOx50 PEtOx100 in black, red and blue respectively
DMA SEC (Figure 35) measurements were performed to determine number average molecular
weight (Mn) and the dispersity Đ. The values were obtained based on calibration with
polymethylmethacrylate (PMMA) standards.
Table 3 : 1H NMR and DMA-SEC results. DMA-SEC values are calibrated with PMMA, 1H NMR spectra were
taken in chloroform-d. Mn is the number average molecular weight, Đ the dispersity and n the number of
repeating units.
Type of polymer
PEtOx20
PEtOx50
PEtOx100
1
H NMR Mn
2300
5000
8700
SEC Mn
3100
7500
15500
SEC Đ
1.08
1.07
1.06
n 1H NMR
23
50
87
n SEC
33
81
165
The low dispersities measured with SEC indicated a well-controlled polymerization (Table
3).53,54 When comparing the values of Mn obtained with 1H NMR spectroscopy and SEC,
different values were obtained with the commonly observed overestimation of Mn for PEtOx
by SEC when calculated against PMMA standards, which have different chemical properties
than PEtOx. The 1H NMR values are most reliable for Mn, as this integration is absolute with
43
respect to the error of integration. The Mn’s of the different polymers were in the intended
range.
Qualitative FT-IR analysis was also taken (Figure 36) as the azide functionality is not visible in
1H
NMR spectroscopy nor SEC. The signal with the highest intensity is the so called Amide I
band, being the stretching of the C=O bond (ν(C=O)).78,79 Other pronounced peaks are the antisymmetric stretch of CH2 at 2940 cm-1, the anti-symmetric stretch of CH3 at 2979 cm-1 and the
symmetric stretch of CH3 and/or CH2 at 2890 cm-1. The most interesting peaks are these of the
azide functionality, which should have a strong asymmetric stretching vibration between 2170
and 2080 cm-1. A weak band between 1345 and 1175 can also be observed due to the
symmetric stretching of N=N=N.78,79 In the FT-IR spectra, this asymmetric stretching is visible
as an intense band at 2101 cm-1 in accordance with wavenumbers for an azide end-group in
PMeOx (poly(2-methyl-2-oxazoline)) reported as 2102 cm-1 54 and 2109 cm-1
53.
Moreover,
while the intensity of the Amide I band of the PEtOx20, PEtOx50 and PEtOx100 stays the same,
the azide asymmetric stretching vibration lowers in intensity with increasing number of
repeating units. This decrease can nicely be attributed to the decrease in concentration of
azide when going from PEtOx20 to PEtOx100. Out of the FR-IR spectrum, it is not possible to
rigorously assign the wavenumber of the azide symmetric stretching band. The band around
1270 might be of the azide functionality as the intensity decreases in the same trend as the
2101 asymmetric stretching vibration band. These results point out the azide functionality is
attached to the polymer as well as confirm the expected decrease in azide content with
increasing polymer molar mass.
MALDI-TOF analysis was taken of all three the polymers (Figure 37 for PEtOx20, SI 10 for
PEtOx50 and PEtOx100). In the zoom (Figure 37, right), the m/z’s of 2260, 2359 and 2458
correspond to the Na+ salts of PEtOx22, PEtOx23 and PEtOx24 respectively, with 99 Da the mass
of the repeating units. Other recurring peaks are around 2233, 2332 and 2431, which are
attributed to the loss of nitrogen from the end-group due to photolysis of the laser (Figure
38), resulting in a nitrene functional group.80,81 Other distinct peaks present in the spectrum
are the K+ salts. Peaks coming from chain-transfer reactions result in a proton-initiated 50 and
chain coupled polymer 50 (Figure 38) and are also present in the MALDI spectrum.
44
Figure 36: FTIR analysis of PEtOx20-N3, PEtOx50-N3, PEtOx100-N3 in black, red and blue respectively.
Measurements were taken on solid samples of the polymers. References 78,79 were used for assignment of the IR
bands.
Figure 37: MALDI-TOF spectrum of PEtOx20. Left the spectrum between 1000 and 3000 Da, right a zoom
between 2200 and 2500 Da.
45
Figure 38: Side products visible in the MALDI-TOF spectrum
2.4 Cu(I)-Catalyzed Azide-Alkyne Cycloadditions
When the synthesis of protoligand 7A and the azide functionalized oligomers and polymers
were prepared, Cu catalyzed azide-alkyne reactions needed to be carried out to yield the
functional ligands. Because time was running short and only a small amount of protoligand 7A
was prepared, the cycloaddition with TEG-azide 13 is the sole Cu(I) AAC carried out.
Figure 39: TEG-azide is attached to the protoligand 7A via Cu(I)AAC
Synthesis of 4,6-bis[4’-((2-(2-(2-methoxyethoxy)ethoxy)ethyl)-1”,2”,3”-triazol-4”-yl)pyrid-2’yl]-2-phenylpyrimidine 8A-TEG was inspired by literature conditions33 and knowledge of our
group. TEG-azide 13 was used with an equivalence of 13 to protoligand 7A. Cu(I) and
N,N,N′,N′′,N′′-pentamethyldiethylenetriamine (PMDETA) were added with equivalence 1.6
and sodium ascorbate with an equivalence of 0.2. The mixture was stirred overnight at room
temperature by which the color of the mixture changed from deep green to deep purple. The
next day, TLC analysis was taken using the same conditions as for the synthesis of the
protoligand. TEG-azide 13 was they only compound moving, all the rest stayed on the baseline,
indicating either the reaction worked or either the protoligand 7A complexated with Cu(I) and
stayed on the baseline. Work up was done by filtering over an aluminum oxide column by first
46
adding THF, to get rid of the Cu(I). First, the filter was washed with THF, followed by DCM and
DCM/MeOH to a 5/1 ratio with a drop of NEt3. Compound 8A-TEG was not obtained, but the
unreacted begin products were. The aluminum oxide still had a greenish color, indicating that
Cu(I) doesn’t come off. The possibility exists that the monocoupled product of 8A and one
TEG-N3 13 or the dicoupled 8A-TEG is still on the aluminum oxide. Because time was running
short, other reaction and workup conditions were not carried out anymore.
47
48
3 Conclusion and Outlook
Synthesis of 2-bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3 was carried out according to
literature conditions. For purifying the compound, a Kugelrohr and flash column
chromatography were used in series. Literature conditions for the synthesis of 2[(triisopropylsilyl)ethynyl]-6-(tributylstannyl)pyridine 4 only yielded compound 9, the
debrominated compound 2-[(triisopropylsilyl)ethynyl]-pyridine (Figure 37). At first, the failure
of the reaction was attributed to impurities in the beginning compound 3 and the
tributyltinchloride. When more pure compounds and more dry reaction environments did not
work either, other reaction conditions were used in which the mixture was hold at lower
temperatures. By this, compound 4 could be obtained.
Figure 37: The major compounds formed in the stannylation reaction
For synthesizing compound 6, no literature conditions were found. Nonetheless, Stille
reactions of compound 4 and Stille couplings of 4-iodopyrimidine have been reported with the
used conditions, but that method could not be translated for synthesizing 6. Possible causes
are the presence of impurities in 4,6-diiodopyrimine 5. 4,6-Dichloro-2-phenylpyrimidine 5A
(fenclorim) on the other hand, which was more pure did work in the Stille coupling with the
stannylated compound 4. By this, 6A was obtained, which is another compound than initially
intended. With prospect to the grid forming ligand, the phenyl group of compound 6A is not
suspected to impede grid formation. Moreover, this phenyl group is thought to stabilize
supramolecular grid formation via π-π stacking.10
Figure 38: Compound 6A was made instead of 6
49
As for the synthesis of azide functionalized attachable oligomers and polymers, 2-(2-(2methoxyethoxy)ethoxy)ethyl azide 13 and PEtOxn-N3 (poly-α-methyl-ω-azido-(2-ethyl-2oxazoline)), with n=20, 50 and 100, were successfully prepared.
Figure 39: 2-(2-(2-Methoxyethoxy)ethoxy)ethyl azide 13 and PEtOxn-N3, with n=20,50 and 100
Compound 13 was obtained with high purity. The PEtOx-N3 polymers were obtained with a
low dispersity as determined by DMA-SEC. The obtained number average molecular weights
were close to the intended values and were analyzed by 1H NMR spectroscopy. The
characterization of the azide end functionality was done by FT-IR and MALDI.
A Cu(I)AAC between protoligand 7A and TEG-N3 13 was carried out. With the used conditions,
ligand 8A-TEG was not obtained due to no optimal reaction conditions and/or workup.
Because time was running short, this reaction could not be studied further.
Figure 40: Cu(I)AAC between protoligand 7A and 2-(2-(2-methoxyethoxy)ethoxy)ethyl azide 13
Because the synthesis of compound 7 took longer than anticipated, unfortunately no studies
on the behavior of the compound regarding supramolecular grids could yet be done, nor could
they be introduced in hydrogels for studying self-healing character. More research is needed
to get answers to those pressing questions.
50
51
52
4 Experimental
4.1 Materials and Equipment
Reagents were bought at Sigma Aldrich. Exceptions are TBAF 1M solution in THF, which was
bought from Fluka. Methanol-d4 was bought from Euriso-top. 2-Ethyl-2-Oxazoline was kindly
donated by Polymer Chemistry Innovations.
Proton nuclear magnetic resonance was recorded on a Bruker Avance 300 MHz at room
temperature. NMR spectra are measured in chloroform-d (CDCl3) or dimethyl sulfoxide-d6
DMSO-d6 (DMSO-d6) bought from Euriso-top. The chemical shifts are given in parts per million
(δ), relative to 7.26 ppm for CHCl3 and 2.54 for DMSO.
Carbon 13 nuclear magnetic resonance was recorded on a Bruker Avance II 500 MHz at room
temperature. NMR spectra are measured in chloroform-d (CDCl3) bought from Euriso-top. The
chemical shifts are given in parts per million (δ), relative to CHCl3 at 77.36 ppm.
Chromatographic columns on aluminum oxide and silica were performed on Merck Alox 90
standard aluminum oxide and on Davisil chromatographic silica mecia LC60A 70-200 micron
respectively.
PD-10 columns were performed on GE Healtcare PD-10 pre-packed columns, SephadexTM G25M.
Silica TLC’s were taken on Macherey-Nagel SILG-25 UV254 glass plates. Aluminium oxide TLC’s
were taken on Merck TLC Aluminum oxide 60 F254 neutral.
Size-exclusion chromatography (SEC) was performed on a Agilent 1260-series HPLC system
equipped with a 1260 online degasser, a 1260 ISO-pump, a 1260 automatic liquid sampler
(ALS), a thermostatted column compartment (TCC) at 50°C equipped with two PLgel 5 µm
mixed-D columns and a mixed-D guard column in series, a 1260 diode array detector (DAD)
and a 1260 refractive index detector (RID). The used eluent was DMA containing 50 mM of
LiCl at a flow rate of 0.500 ml/min. The spectra were analyzed using the Agilent Chemstation
software with the GPC add on. Molar mass and PDI values were calculated against PMMA
standards from PSS.
53
A solvent purification system that is used in combination with the glove box is from Meyer,
custom made, and has got a nitrogen, aluminum oxide drying system. The Vigor glovebox has
a system for automatic oxygen and water removal. Oxygen and water values are below 1 ppm
and 0.1 ppm respectively. An automatic solvent scrubber is installed. Purified solvents were
purified with the solvent purification system and directly used.
A Martin Christ Alpha 2-4 LDPlus with an ice condenser capacity of 4 kg and temperature of 85°C and 4kg/24h performance was used as freezedryer.
A Büchi GRK50 Kugelrohr was used together with a Thyracont VD83 manometer.
All FT-IR spectra were measured using PerkinElmer Frontier FT-IR (midIR) combined with a
MKII Golden Gate set-up equipped with a diamond crystal from Specac. The spectra were
measured with a wavenumber window between 4000 and 600 cm-1. The material was placed
on the measuring plate and the crystal was brought into contact with the dry powder. After
measurement was complete, the plate and the crystal were cleaned with isopropanol.
PerkinElmer Spectrum Analysis software was used to analyze the results.
LCMS analysis was performed on an Agilent 1100 HPLC with a quaternary pump and UV-DAD
detection, coupled with an Agilent G1956B MSD and an ESI ionization source. The used column
was a Phenomenex - Kinetex C18 (5 µm 150x4.6 mm). A flowrate of 1.5 ml/min was used with
a temperature of 35°C and an injection volume of 15 µl. Solvent A was 5 mM NH4OAc in H2O
whereas solvent B was ACN. These solvent were used in a gradient in 6 minutes. Gradients of
0100, 75100 and 90100 were used, always ending in 100% of B. The mass-range was
80-1000. The positive and negative ions are measured separately.
ESI-MS analysis was performed on an Agilent 1100 HPLC with a quaternary pump and UV-DAD
detection, coupled with an Agilent G1956B MSD and an ESI ionization source. The samples
were injected by direct injection and 5 µl samples are loaded. Solvent A was 5 mM NH4OAc in
H2O whereas solvent B was ACN. A 50/50 ratio of the two samples were used. The mass-range
was 80-3000. The positive and negative ions are measured separately.
GCMS analysis was performed on an Agilent 6890 GC coupled to the 5973 MSD with EI
ionization source. The used column was a DB-5ms Agilent J&W Scientific with a 5%-Phenyldimethylpolysiloxane phase. This is a non-polar column with a length of 60 m and an ID of 0.25
54
mm and 0.25 µm film. The maximum allowable operating temperature (MAOT) is 325°C. A
column flow of 1.3 mL/min in constant flow mode was used. The septum purge was 2 mL/min
with a split vent which was optimal at 20 ml/min. Electronic pressure control for the head
pressure was used. The carried gas used was He. An inlet temperature of 250°C was used with
a heated transfer line at 280°C. The used temperature gradient was 3 min at 70°C, 17.5°C/min
to 320°C, 12.71 min 320°C. In total, this is 30 min. 4.8 min solvent delay time was used,
together with 2 min split less injection mode. The MSD mass range is 10-800 amu and was
measured from 10 to 400 (MS400) or 10 to 800 (MS800).
Matrix assisted laser desorption/ionization time of flight mass spectroscopy (MALDI-TOF MS)
was performed on an Applied Biosystems Voyager. The STR MALDI-TOF mass spectrometer
equipped with 2 m linear and 3 m reflector flight tubes and a 355 nm Blue Lion Biotech
Marathon solid state laser (3.5 ns pulse). All mass spectra were obtained with an accelerating
potential of 20 kV in positive ion mode and in either reflectron or linear mode. Trans-2-[3-(4tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile
(DCTB)
or
2-(4’-Hydroxy-
benzeneazo)benzoic acid (HABA) (20 mg/ml in acetone) were used as matrix and NaTFA (2
mg/ml in acetone) was used as cationizing agent. Polymer samples were dissolved in acetone
(2 mg/ml). Analyte solutions were prepared by mixing 10 µl of the matrix solution and 5 µl of
the polymer solution, with or without 5 µl of the salt solution. Subsequently, 0.5 µl of this
mixture was spotted on the sample plate, and the spots were dried in air at room temperature.
A poly(ethylene oxide) standard (Mn = 2000 g/mol) was used for calibration. All data was
processed using the Data Explorer 4.0.0.0 (Applied Biosystems) software package.
4.2 Ligand Synthesis
4.2.1 2-Bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3, PdCl2(PPh3)2 Catalyst,
Purification by Column Chromatography
2,6-Dibromopyridine (330 mg, 1.39 mmol), PdCl2(PPh3)2 (22 mg, 0.0313 mmol) and CuI (12.6
mg, 0.0662 mmol) were dissolved in dry THF (3 ml), together with NEt3 (0.3 ml). This mixture
was bubbled with Ar for half an hour. (Triisopropylsilyl)acetylene (0.10 ml, 0.45 mmol) was
added dropwise using a syringe and the mixture is stirred for four hours at 40°C. After the
reaction, the mixture was filtered and the solvent evaporated. The product was purified by
55
column chromatography on silica. The silica was first run with n-hexane/NEt3 99.5/5 and
subsequently with n-hexane before loading the crude. n-hexane and EtAc are used as eluents
starting from 0% to 4% of EtAc. The column is followed by TLC on silica with n-hexane/EtAc
85/15%. The middle of the three spots is the right product (rf = 0.73). The fraction containing
most
of
the
middle
spot
is
run
again
on
a
same
column.
2-Bromo-6-
[(triisopropylsilyl)ethynyl]pyridine was obtained as a colorless oil with a yield of 11 % (3.60
mg, 0.0107 mmol).
1H
NMR, 300 MHz, CDCl3: 7.51-7.39 (m, 3H, pyrimidine-H), 1.13 (s, 18H, –Si-(CH-(CH3)2)3), 1.12
(s, 3H, –Si-(CH-(CH3)2)3)
13C
NMR, 500 MHz, CDCl3: 143.8 (-CH=C-CΞC-TIPS); 141.6(=N-CBr=C-); 138.1 (-CH-CH=C-CΞC-
TIPS); 127.6 (=N-CBr=C-); 126.8 (-CH=C-CΞC-TIPS); 104.4 (=C-CΞC-TIPS-); 93.9 (=C-CΞC-TIPS);
18.6 (–Si-(CH-(CH3)2)3); 11.2(–Si-(CH-(CH3)2)3)
ESI-LCMS: [M+H]+ = 340.1
4.2.2 2-Bromo-6-[(triisopropylsilyl)ethynyl]pyridine
3,
Pd(PPh3)4
Catalyst,
Purification by Column Chromatography
2,6-Dibromopyridine (337 mg, 1.42 mmol), PdCl2(PPh3)2 (32.10 mg, 0.02778 mmol) and CuI
(15.75 mg, 0.08270 mmol) were dissolved in dry THF (3 ml), together with NEt3 (0.3 ml). This
mixture was bubbled with Ar for half an hour. (Triisopropylsilyl)acetylene ( 0.10 ml, 0.45 mmol)
was added dropwise using a syringe and the mixture was stirred for four hours at 40°C. After
the reaction, the mixture was filtered and the solvent evaporated. The product was purified
by column chromatography on silica. The silica is first run with n-hexane/NEt3 99.5/0.5 and
subsequently run with n-hexane before loading of the crude. n-Hexane and EtAc are used as
eluents starting from 0% to 4% of EtAc. The column is followed by TLC on silica with nhexane/EtAc 85/15%. The middle of the three spots is the right product (rf = 0.73). The fraction
containing most of the middle spot is run again on a similar column. 2-bromo-6[(triisopropylsilyl)ethynyl]pyridine was obtained as a colorless oil with a yield of 10 % (3.13
mg, 0.00925 mmol).
56
1H
NMR, 300 MHz, CDCl3: 7.51-7.39 (m, 3H, pyrimidine-H), 1.13 (s, 18H, –Si-(CH-(CH3)2)3), 1.12
(s, 3H, –Si-(CH-(CH3)2)3)
13C
NMR, 500 MHz, CDCl3: 143.8 (-CH=C-CΞC-TIPS); 141.6(=N-CBr=C-); 138.1 (-CH-CH=C-CΞC-
TIPS); 127.6 (=N-CBr=C-); 126.8 (-CH=C-CΞC-TIPS); 104.4 (=C-CΞC-TIPS-); 93.9 (=C-CΞC-TIPS);
18.6 (–Si-(CH-(CH3)2)3); 11.2(–Si-(CH-(CH3)2)3)
ESI-LCMS: [M+H]+ = 340.1
4.2.3 2-Bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3, Purification by Kugelrohr
2,6-Dibromopyridine (7759.9 mg, 32.76 mmol), PdCl2(PPh3)2 (522 mg, 0.7435 mmol) and CuI
(306 mg, 1.607 mmol) were loaded in a schlenk flask. Using a schlenk line, the schlenk flask
was put under vacuum followed by argon three times. The powders were dissolved in dry THF
(70 ml) with NEt3 (8 ml), which was added using a syringe. (Triisopropylsilyl)acetylene (2.5 ml,
11.14 mmol) was added dropwise using a syringe and the mixture is stirred for two hours at
40°C. After the reaction, the mixture was filtered and the solvent evaporated. The crude was
loaded in a 25 ml round bottom flask by dissolving it in DCM. This round bottom flask is put in
the Kugelrohr and run at 80-90 °C by a pressure of 1.0-0.4 mbar (see below for the Kugelrohr
setup). The destillation is followed by TLC on silica with 85/15 n-hexane/EtAc. The distillation
is stopped when the spot with rf = 0.5 is gone. The content in the Kugelrohr which was not
distilled over was purified by flash column chromatography on silica with n-hexane/EtAc 7/3.
2-Bromo-6-[(triisopropylsilyl)ethynyl]pyridine was obtained as a colorless oil with a yield of 68
% (2572,6 mg, 7,6031 mmol).
1H
NMR, 300 MHz, CDCl3: 7.51-7.39 (m, 3H, pyrimidine-H), 1.13 (s, 18H, –Si-(CH-(CH3)2)3), 1.12
(s, 3H, –Si-(CH-(CH3)2)3)
13C
NMR, 500 MHz, CDCl3: 143.8 (-CH=C-CΞC-TIPS); 141.6(=N-CBr=C-); 138.1 (-CH-CH=C-CΞC-
TIPS); 127.6 (=N-CBr=C-); 126.8 (-CH=C-CΞC-TIPS); 104.4 (=C-CΞC-TIPS-); 93.9 (=C-CΞC-TIPS);
18.6 (–Si-(CH-(CH3)2)3); 11.2(–Si-(CH-(CH3)2)3)
ESI-LCMS: [M+H]+ = 340.1
57
Kugelrohr setup: A Kugelrohr setup, with a rotation oven was used for purifying the crude. The
oven of the Kugelrohr used in the lab was able to get to a temperature of 250°C. A round
bottom flask is loaded in the oven and while it is spun, is heated. At the other end, outside the
oven, an oil pump is connected, to lower the pressure inside the round bottom flask.
Immediately outside the oven, a spat bowl is used to collect the compound which is distilled
out of the oven. The setup is equipped with a manometer. Right after the collecting spat bowl,
a tap is placed to control the vacuum.
4.2.4 2-[(Triisopropylsilyl)ethynyl]-6-(tributylstannyl)pyridine 4
2-Bromo-6-[(triisopropylsilyl)ethynyl]pyridine (96 mg, 0.28 mmol) was dissolved in dry THF (7
ml) and stirred under argon atmosphere. The solution was cooled down to -78°C.
Subsequently, n-BuLi (2.5 M in hexane, 0.14 ml, 0.34 mmol) was added dropwise after which
the mixture was allowed to heat till -20°C. After 45 minutes, the vessel was cooled down to 78°C again. A solution of Bu3SnCl (0.11 ml, 0.39 mmol) in THF (4 ml) was added dropwise. The
reaction was allowed to warm to ambient temperature and stirred overnight. 2[(Triisopropylsilyl)ethynyl]-6-(tributylstannyl)pyridine was not obtained.
4.2.5 2-[(Triisopropylsilyl)ethynyl]-6-(deuterio)pyridine Test Reaction
2-Bromo-6-[(triisopropylsilyl)ethynyl]pyridine (30 mg, 0.089 mmol) was dissolved in dry THF
(2 ml) and stirred under argon atmosphere. The solution was cooled down to -78°C.
Subsequently, n-BuLi (2,5 M in hexane, 0.05 ml, 0.12 mmol) was added dropwise.
Subsequently, the mixture was allowed to heat till -20°C. After 45 minutes, the vessel was
cooled down to -78°C again. MeOD (0.01 ml, 0.24 mmol) was added. The reaction was allowed
to warm to ambient temperature and stirred overnight. 2-[(Triisopropylsilyl)ethynyl]-6-(
deuterio)pyridine was found and was not isolated.
1H
NMR, 300 MHz, CDCl3: 7.63 (t, J=7.73, 1H, D-C-CH=CH), 7.46 (dd, J1=3.72, J2=5.30, 1H, D-C-
CH=CH), 7.21 (d, J=7.57, 1H, -CH=C-CΞC-TIPS), 1.15 (s, 18H, –Si-(CH-(CH3)2)3), 1.14 (s, 3H, –Si(CH-(CH3)2)3)
ESI-LCMS: [M+H]+ = 261.1
58
4.2.6 2-[(Triisopropylsilyl)ethynyl]-6-(tributylstannyl)pyridine 4
2-Bromo-6-[(triisopropylsilyl)ethynyl]pyridine (1010 mg, 2.988 mmol) was dissolved in THF (70
ml) and stirred under argon atmosphere. The solution was cooled down to -78°C.
Subsequently, n-BuLi (1.4 ml, 2.5 M solution in n-hexane, 3.5 mmol) was added dropwise.
After stirring the mixture for 45 minutes at -78°C, a solution of newly bought Bu3SnCl (1.1 ml,
4.1 mmol) in THF (10 ml) was added dropwise. The reaction mixture was stirred at -78°C. After
six hours, the mixture was allowed to warm to room temperature and stirred overnight. The
solvents were evaporated and the residue was purified by column chromatography on
alumina with n-pentane as eluent. 2-[(Triisopropylsilyl)ethynyl]-6-(tributylstannyl)pyridine
was obtained as a colorless oil (476 mg, 0,868 mmol) with a yield of 29%.
1H
NMR, 300 MHz, CDCl3: 7.40 (dd, J1=6.74, J2=8.38, 1H, Bu3Sn-C-CH=CH-), 7.28 (d, J=7.28, 1H,
Sn-C-CH=CH), 7.27 (d, J=7.28, 1H, CH=C-CΞC-TIPS),1.56 (m, 6H, CH3-CH2-CH2-CH2-Sn-), 1.33 (s,
J=7.5Hz, 6H, CH3-CH2-CH2-CH2-Sn-), 1.15 (s, 18H, –Si-(CH-(CH3)2)3), 1.14 (s, 3H, –Si-(CH(CH3)2)3), 1.10 (m, 6H, CH3-CH2-CH2-CH2-Sn-), 0.88 (t, J=7.27, 9H, CH3-CH2-CH2-CH2-Sn-)
13C
NMR, 500 MHz, CDCl3: 174.6 (Bu3Sn-C=C-); 144.1 (-CH=C-CΞC-TIPS); 132.6 (Bu3Sn-C=C-C=);
130.9 (Bu3Sn-C=C-); 126.1 (-CH=C-CΞC-TIPS); 107.4 (=C-CΞC-TIPS); 90.5 (=C-CΞC-TIPS); 29.0
(CH3-CH2-CH2-CH2-Sn-); 27.0 (CH3-CH2-CH2-CH2-Sn-); 18.7 (–Si-(CH-(CH3)2)3); 13.7 (–Si-(CH(CH3)2)3); 11.4 (CH3-CH2-CH2-CH2-Sn-); 10.1 (CH3-CH2-CH2-CH2-Sn-)
ESI-LCMS: [M+H]+ = 260.1
4.2.7 Stille Coupling Diiodopyrimidine 6
2-[(Triisopropylsilyl)ethynyl]-6-(tributylstannyl)pyridine 4 (400 mg, 0.729 mmol), 4,6diiodopyrimidine 5 (98.3 mg, 296 mmol) and Pd(PPh3)4 (43.2 mg, 3.74 µmol) were brought in
a dry round bottom flask and distilled toluene (30 ml) was added. The mixture was stirred
under argon atmosphere at 80°C for 8 days. The solvent was evaporated and a black oil with
black precipitate is obtained. A column chromatography on silica with DCM with 1% of NEt3
was performed. In the end, till 10% of MeOH was added to the eluents. The silica was first run
with DCM and 1% of NEt3 and the column is followed by TLC with n-pentane/DCM 85/15 and
59
a drop of NEt3. The desired 4,6-Bis[4’-((triisopropylsilyl)ethynyl)pyrid-2’-yl]-2-pyrimidine 6
was not obtained.
4.2.8 4,6-Bis[4’-((triisopropylsilyl)ethynyl)pyrid-2’-yl]-2-phenylpyrimidine
6A
with PdCl2(PPh3)2
2-[(Triisopropylsilyl)ethynyl]-6-(tributylstannyl)pyridine 4 (43 mg, 0.078 mmol), 4,6-dichloro2-phenylpyrimidine 5A (8,2 mg, 36 mmol) and PdCl2(PPh3)2 (2.8 mg, 3.9 µmol) were brought
in a dry round bottom flask and dry DMF (3.5 ml) was added. The mixture was stirred under
argon atmosphere at 80°C for 42 hours. The solvent was evaporated and a brown oil is
obtained. A column chromatography on silica with n-pentane/DCM (starting at 100/0 to
70/30) with a drop of NEt3 was performed. The silica was first run with n-pentane and a drop
of NEt3. The column is followed by TLC with n-pentane/DCM 85/15 and a drop of NEt3. The
fraction containing the second spot is then recrystallized from EtOH, starting from EtOH under
reflux and letting it slowly cool down to get the white crystalline 4,6-bis[4’((triisopropylsilyl)ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 6A with a yield of 57% ( 14 mg,
0.021 mmol).
1H
NMR, 300 MHz, CDCl3: 9.30 (s, 1H, -N=C(-pyridine)-CH=C(-pyridine)-N=); 8.72-8.69 (m, 2H,
=CH-C(-pyrimidine)=CH-); 8.66 (dd, J1=1.05, J2=7.93, 2H, -N=(pyrimidine-)C-CH=CH- CH=C-CΞCTIPS); 7.85 (t, J=7.82, 2H, -N=(pyrimidine-)C-CH=CH-CH=C-CΞC-TIPS); 7.61 (dd, J1=1.06, J2=7.70,
2H, -N=(pyrimidine-)C-CH=CH-CH=C-CΞC-TIPS); 7.59-7.52 (m, 3H, -CH=CH-CH=CH-C(pyrimidine)=CH-); 1.20 (s, 36H, –Si-(CH-(CH3)2)3); 1.19 (s, 6H, –Si-(CH-(CH3)2)3)
ESI-MS: [M+H]+= 671.4
4.2.9 4,6-Bis[4’-((triisopropylsilyl)ethynyl)pyrid-2’-yl]-2-phenylpyrimidine
6A
with Tetrakis
2-[(Triisopropylsilyl)ethynyl]-6-(tributylstannyl)pyridine 4 (43 mg, 0.078 mmol), 4,6-dichloro2-phenylpyrimidine 5A (8.2 mg, 36 mmol) and tetrakis (4.5 mg, 3.9 µmol) were brought in a
dry round bottom flask and dry DMF (3.5 ml) was added. The mixture was stirred under argon
atmosphere at 80°C for 42 hours. The solvent was evaporated and a brown oil is obtained. A
60
column chromatography on silica with n-pentane/DCM (starting at 100/0 to 70/30) with a
drop of NEt3 was performed. The silica was first run with n-pentane and a drop of NEt3. The
column is followed by TLC with n-pentane/DCM 85/15 and a drop of NEt3. The fraction
containing the second spot is then recrystallized in with EtOH, starting from EtOH under reflux
and
letting
it
slowly
cool
down
to
get
the
white
crystalline
4,6-bis[4’-
((triisopropylsilyl)ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 6A with a yield of 57% (14 mg, 0.021
mmol).
1H
NMR, 300 MHz, CDCl3: 9.30 (s, 1H, -N=C(-pyridine)-CH=C(-pyridine)-N=); 8.72-8.69 (m, 2H,
=CH-C(-pyrimidine)=CH-); 8.66 (dd, J1=1.05, J2=7.93, 2H, -N=(pyrimidine-)C-CH=CH- CH=C-CΞCTIPS); 7.85 (t, J=7.82, 2H, -N=(pyrimidine-)C-CH=CH-CH=C-CΞC-TIPS); 7.61 (dd, J1=1.06, J2=7.70,
2H, -N=(pyrimidine-)C-CH=CH-CH=C-CΞC-TIPS); 7.59-7.52 (m, 3H, -CH=CH-CH=CH-C(pyrimidine)=CH-); 1.20 (s, 36H, –Si-(CH-(CH3)2)3); 1.19 (s, 6H, –Si-(CH-(CH3)2)3)
ESI-MS: [M+H]+= 671.4
4.2.10 4,6-Bis[4’-(ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 7A
4,6-Bis[4’-((triisopropylsilyl)ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 6A (27 mg, 0.0402 mmol)
was dissolved in dry THF (4 ml) in a round bottom flask and stirred. TBAF (1M solution in THF,
0.15 ml, 0.15 mmol) was added dropwise. The solvent is evaporated and thereafter, water is
added. Chloroform is added and the chloroform phase is extracted 3 times more with water.
The chloroform phase is evaporated and a brown oil is obtained. The oil is purified by a column
chromatography on silica with n-pentane/DCM/MeOH in a gradient starting from 85/15/0 to
0/90/10 with 0.5% of NEt3. Afterwards, the product was dissolved in CHCl3 and washed with
water to get rid of the NEt3. 4,6-Bis[4’-(ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 7A was
obtained as a yellow solid with a yield of 97% (14 mg, 0.038 mmol).
1H
NMR, 300 MHz, CDCl3: 9.29 (s, 1H, -N=C(-pyridine)-CH=C(-pyridine)-N=); 8.72-8.69 (m, 4H,
=CH-C(-pyrimidine)=CH-, -N=(pyrimidine-)C-CH=CH-CH=C-CΞC-TIPS); 7.89 (t, J=7.83, 2H, N=(pyrimidine-)C-CH=CH-CH=C-CΞC-TIPS); 7.62 (dd, J1=1.06, J2=7.70, 2H, -N=(pyrimidine-)CCH=CH-CH=C-CΞC-TIPS); 7.59-7.51 (m, 3H, -CH=CH-CH=CH-C(-pyrimidine)=CH-); 3.25 (s, 2H, HCΞC-)
61
ESI-MS: [M+H]+ = 359.2
4.3 Polymer and Oligomer Synthesis
4.3.1 2-(2-(2-Methoxyethoxy)ethoxy)ethyl Azide
4.3.1.1 2-(2-(2-Methoxyethoxy)ethoxy)ethyl Tosylate 12
Triethylene glycol monomethyl ether (9.7 ml, 61 mmol), triethylamine (21.2 ml, 0.1525 mol)
and 4-dimethylaminopyridine (DMAP; 1.74 g, 14.2 mmol) were dissolved in 400 ml of DCM.
The mixture was cooled to 0°C and 4-toluenesulfonylchloride (13.2 g, 69 mmol) was added.
After some minutes, the solution got an orange color and the ice bad was removed and the
mixture was stirred overnight at room temperature. The solution was washed with water and
dried over MgSO4. After evaporation of the solvent, column chromatography was used to
purify the compound with silica as stationary phase and EtAc/DCM 3/7 as the eluent. 2-(2-(2Methoxyethoxy)ethoxy)ethyl tosylate was obtained as a yellow oil (13.45 g, 42.3 mmol, 70
%).
1H
NMR, 300 MHz, CDCl3: 7.77 (d, J=8.3 Hz, 2H, RO3S-C-CH=CH-), 7.33 (d, J=8.0, 2H, RO3S-C-
CH=CH-), 4.41 (m,2H, Tos-CH2-), 3.65 (m, 2H, Tos-CH2-CH2-O-), 3.56 (m, 6H, CH3-O-CH2-CH2-OCH2-) 3.51 (m, 2H), 3.34 (s, 3H, CH3-O-CH2-), 2.41 (s, 3H, RO3S-C-CH=CH-C-CH3)
ESI-LCMS: [M+NH4]+=336.2
4.3.1.2 2-(2-(2-Methoxyethoxy)ethoxy)ethyl Azide 13
2-(2-(2-Methoxyethoxy)ethoxy)ethyl tosylate (3.0 g, 9.4 mol) was dissolved in 20 ml of purified
DMF under argon. NaN3 (2,11 g, 32 mmol) was slowly added with a plastic spoon. The reaction
was stirred at 60°C. After 48 hours, 60 ml water was added to the mixture. An extraction was
performed with diethylether. The organic phase was dried with MgSO4, filtered and dried
under vacuum. A yellow oil was obtained (1.5 g, 7.9 mmol, 84%). The compound was stored
in solution with Et2O in the freezer for safety.
62
1H
NMR, 300 MHz, CDCl3: 3.64 (m, 8H, CH3-O-CH2-CH2-O-CH2- CH2-O-), 3.52 (m, 2H, -O-CH2-
CH2-N3), 3.36 (m, 5H, CH3-O-CH2-CH2-O-CH2- CH2-O-CH2-CH2-N3)
ESI-MS: [M+H]+=190.2 ; [M+NH4]+= 207.2
4.3.2 Poly-α-methyl-ω-azido-(2-ethyl-2-oxazoline)
4.3.2.1 Desalting Protocol with PD-10
Standard procedure:
The polymer and salt mixture is dissolved in water. 2.5 ml of the solution is brought on the PD10 column. When the solution has entered the column completely, 3.5 ml of water is used to
elute this first fraction. This fraction contains most of the polymer. Afterwards, 25 ml of water
is added and kept as a second fraction. This is the salt fraction. After having desalted all the
polymer/salt solution a first time, all first fractions were desalted a second time following the
same procedure.
Non-standard procedure:
The polymer and salt mixtures is dissolved in water. 2.5 ml of the solution is brought on
column. When the solution has entered the column completely, 4.5 ml of water is used to
elute this first fraction. This fraction contains most of the polymer and a reasonable amount
of water. Afterwards, 25 ml of water is added and kept as a second fraction. This is the salt
fraction. After having desalted all the polymer/salt solution a first time, all first fractions were
desalted a second time following the same procedure. After the second desalting condition,
the first fraction was purified a third time. This third time, 4 ml is used as eluent volume.
4.3.2.2 PEtOx20-N3
All three the PEtOx polymers were characterized by 1H NMR, DMA-SEC and MALDI-TOF. The
DMA-SEC and MADI-TOF results are added in 2.3.2 Synthesis of PEtOx-N3 in Figures 35 and 37
and in SI 10.
63
EtOx (1.211 ml ,12.0 mmol) and MeOTs (90.84 µl ,0.652 mmol) were brought in a round
bottom flask in a glove box and dissolved in ACN (1.789 ml) and stirred at 100°C for 25 minutes.
Subsequently, NaN3 (0.117 g, 1.80 mmol) was added. The mixture was taken out of the
glovebox and the solvent was evaporated. Water was added and the solution was purified on
a PD-10 desalting column using the non-standard procedure. After freeze drying, 0.3984 mg
of PEtOx20-N3 was obtained (40% yield).
1H
NMR, 300 MHz, CDCl3: 0.96-1.20 (300 H, CH3-[N((C=O)-CH2-CH3)-CH2-CH2]-N3); 2.25-2.50
(200H, CH3-[N((C=O)-CH2-CH3)-CH2-CH2]-N3); 3.00 (13,2 H, CH3-[N((C=O)-CH2-CH3)-CH2-CH2]N3); 3.43 (400 H, CH3-[N((C=O)-CH2-CH3)-CH2-CH2]-N3)
DMA-SEC: Mn = 3100; Đ = 1.08
4.3.2.3 PEtOx50-N3
EtOx (1.211 ml ,12.0 mmol) and MeOTs (36.3 µl, 0.241 mmol) were brought in a round bottom
flask in a glove box and dissolved in ACN (1.789 ml) and stirred at 100°C for 65 minutes.
Subsequently, NaN3 (0.0470 g, 0.730 mmol) was added. The mixture was taken out of the
glovebox and the solvent was evaporated. Water was added and the solution was purified on
a PD-10 desalting column following the non-standard procedure. After freeze drying, 0.6148
mg of PEtOx20-N3 was obtained (52% yield).
1H
NMR, 300 MHz, CDCl3: 0.96-1.20 (300 H, CH3-[N((C=O)-CH2-CH3)-CH2-CH2]-N3); 2.25-2.50
(200H, CH3-[N((C=O)-CH2-CH3)-CH2-CH2]-N3); 3.00 (6,0 H, CH3-[N((C=O)-CH2-CH3)-CH2-CH2]N3); 3.43 (400 H, CH3-[N((C=O)-CH2-CH3)-CH2-CH2]-N3)
DMA-SEC: Mn = 7500; Đ = 1.07
4.3.2.4 PEtOx100-N3
1.211 ml (12.0 mmol) EtOx and 8.6 µl (0.057 mmol) MeOTs were put in a round bottom flask
in a glove box and dissolved in ACN (1.789 ml) and stirred at 100°C for 120 minutes.
Subsequently, NaN3 (0.0234 g, 0.360 mmol) was added. The mixture was taken out of the
glovebox and the solvent was evaporated. Water was added and the solution was purified on
64
a PD-10 desalting column following the standard procedure. After freeze drying, 0.7861 mg of
PEtOx20-N3 was obtained (66% yield).
1H
NMR, 300 MHz, CDCl3: 0.96-1.20 (300 H, CH3-[N((C=O)-CH2-CH3)-CH2-CH2]-N3); 2.25-2.50
(200H, CH3-[N((C=O)-CH2-CH3)-CH2-CH2]-N3); 3.00 (3,3 H, CH3-[N((C=O)-CH2-CH3)-CH2-CH2]N3); 3.43 (400 H, CH3-[N((C=O)-CH2-CH3)-CH2-CH2]-N3)
DMA-SEC: Mn = 8700; Đ = 1.06
4.4 Cu(I)-Catalyzed Azide-Alkyne Cycloadditions
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
4,6-Bis[4’-(ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 7A (13.7 mg, 38.2 µmol), 2-(2-(2methoxyethoxy)ethoxy)ethyl azide 13 (95.2 mg; 502 µmol) and Na ascorbate (1.87 mg, 9.44
µmol) are brought in a microwave vessel. A dark green solution of CuBr (12.0 mg, 83.7 µmol)
and PMDETA (14.5 mg, 83.7 µmol) in 12 ml of dry DMF is added. The mixture is stirred
overnight under Ar. The next day, the mixture is deep purple. 12 ml of THF is added and the
mixture is filtered over Aluminum Oxide. The Aluminum Oxide is run with THF. Subsequently,
DCM is used to get everything of the aluminum oxide. A green band is visible at the top of the
aluminum oxide. A Second fraction is taken when eluting with 20% MeOH and a couple drops
of NEt3. The green band of Cu now also elutes out of the column. Afterwards, the solvent is
evaporated. 4,6-Bis[4’-((2-(2-(2-methoxyethoxy)ethoxy)ethyl)-1”,2”,3”-triazol-4”-yl)pyrid-2’yl]-2-phenylpyrimidine 8A-TEG is not obtained.
65
66
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74
6
Scientific Article
Supramolecular Grid Structures For Hydrogels With Self-Healing Character
R. Lenaertsa, K. Ryskulovaa, L. Voorhaara, R. Hoogenbooma*
a
Supramolecular Chemistry group, Ghent University, Department of Organic and
Macromolecular Chemistry, Ghent 9000, Belgium
*[email protected]
A new ligand for the formation of supramolecular gridstructures is
synthesized. Different oligomers and polymers can be coupled to the
ligand via a Copper(I) mediated azide alkyne cycloaddition (CuAAC) to
form the functional grid, yielding a supramolecular star polymer. The
metal ions used for grid formation, Fe(II) and Zn(II), are octahedral
coordinating ions which can be used in water and are biocompatible,
which opens up possibilities for biomedical applications.
Keywords
Supramolecular Grid Structures, Cu(I)-Catalyzed Azide-Alkyne Cycloaddition, Poly(2oxazoline)s
Introduction
In this article, the synthesis of a ligand designed for the formation of supramolecular grid
structures (Figure 1) is discussed. This ligand has the useful ability to coordinate with
octahedral metal ions, like Fe(II) and Zn(II), giving it the ability to be used in water, which
opens the door to biomedical applications. Polymers can be attached to the ligand via Cu(I)catalyzed Azide-Alkyne Cycloaddition, with the purpose of making star polymers.
Supramolecular interactions. Supramolecular gridstructures are formed by supramolecular
interactions, examples of which are host-guest interactions, metal-ligand binding, hydrogen
bonding, electrostatic interactions, π-π stacking and hydrophobic effects. These interactions can
be directional or non-directional, and each have different binding strengths, with metal-ligand
complexes being some of the strongest (1). Non-covalent interactions are generally dynamic,
resulting in an equilibrium between the molecules present as a supramolecular assembled entity
and the molecules that are not in this associated assembly (2)-(4). Figure 2 shows a schematic
view of a supramolecular gridstructure with the ligands represented by grey bars. The metalligand interaction is the most important for forming such gridstructures.
Supramolecular grid structures. 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.’ (5) 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. The ligand designed here contains one pyrimidine group,
which is substituted by two pyridine aromatic groups that are on their turn substituted by a
1,2,3-triazole ring (Figure 1). The phenyl in the middle of ligand 8A is designed to get in the
middle of the supramolecular grid, giving stabilizing π-π interactions. (6) The triazole ring is
75
synthesized out of an alkyne functional group. By Cu(I)AAC, different azide functionalized
oligomers and polymers can be attached onto the ligand precursor, yielding a ligand able of grid
formation. Supramolecular star shaped polymers can be made via this method. The designed
grid is based on analogues from literature, with the difference that in literature pyridines are
used instead of the two triazole rings (6). Triazole rings are also reported in terpyridines as
pyridine analogues (7),(8). Fe(II) and Zn(II) are two octahedral coordinating metals and will be
ligated by the sites as shown in Figure 1 by ligation with the nitrogen free electron pairs. The
grid is designed to be used in water. The used R groups are triethylene glycol and poly(2oxazoline)s (PAOx) with lengths of 20, 50 and 100 repeating units, which can be attached to
compound 7A via CuAAC (Figure 3). With these different lengths, it can be studied if the grid
is formed with Fe(II) and Zn(II) when the R groups are polymers.
Figure 1. Left: Structure of ligand 8A showing the sites where iron can be complexated. Right:
PAOx. The R group can be changed by the choice of monomer. I and T are the initiator and
terminator, respectively.
Poly(2-oxazoline)s. The concept of using triazoles as coordination group to get
supramolecular gridstructures is inspired by literature. Triazole analogues of 3,6-di(2pyridyl)pyridazine (DPP) (9) which are reported to form grids by ligation of Cu(I). Two
problems arise when using DPP Cu(I) grids in water. A first problem addressed with DPP Cu(I)
grids is that disproportionation reactions of Cu(I) occur when used in water (10), making water
a bad solvent. Second, copper is toxic for humans, limiting the use in biomedical applications
(11). The ligand designed in this article is thought to be not prone to these limitations.
Figure 2. Concept of using ligand 8 to get gridstructures.
The polymer chosen to obtain the grid forming ligand is poly(2-ethyl-2-oxazoline) (PEtOx).
Poly(2-oxazoline)s (PAOx) can be made by cationic ring opening polymerization of 2oxazolines. In PAOx the R group (Figure 1) can be altered and chosen for the desired properties
(12),(13). It can be a simple alkyl chain like a methyl or an ethyl which results in a hydrophilic
polymer. n-Hexyl as R group on the other hand yields a hydrophobic polymer (12). Besides
simple alkyls, also chemically more interesting moieties can be introduced on this place,
including functional handles like alkenes and esters (13). PAOx are not only interesting because
76
the polymerization of 2-oxazolines is living, the obtained polymers have many applications in
life sciences. Interesting is the stealth behavior of them, meaning they are not recognized as
foreign by the body (14), making them useful for drug and gene delivery (13). PAOx is also
ideal for making hydrogels for drug delivery and as cell culture scaffolds (14). By choosing the
right monomers, hydrophobicity and other properties of the biocompatible hydrogels can be
altered. Surface modifications for making non-fouling surfaces are another biomedically
focused application of PAOx (13). All these interesting features make PAOx a good choice for
attaching to the supramolecular grid.
Experimental
The synthesis of the compounds is described in the Supporting Information.
Results and Discussion
First, the synthesis of compound 7A (Figure 3) is discussed. The preparation of mono azide
functionalized triethyleneglycol (TEG) and poly-α-methyl-ω-azido-(2-ethyl-2-oxazoline)
(PEtOxN3) with 20, 50 and 100 repeating units is clarified subsequently. In the last part, the
cycloaddition between the TEG and protoligand 7A to obtain functional ligand 8A-TEG is
explained.
Synthesis of protoligand 7A
Figure 3. Synthesis route for obtaining compound 8. The designed R groups are triethylene
glycol and poly(2-oxazoline)s with lengths of 20, 50 and 100 repeating units.
The synthesis of 2-bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3 involves the asymmetric
substitution of only one bromine atom of the 2,6-dibromopyridine 1 by a Sonogashira coupling
reaction (Figure 17). PdCl2(PPh3)2 and Pd(PPh3)4 (tetrakis) were both tested as catalyst, of
which PdCl2(PPh3)2 is the catalyst used in literature (15). The reaction worked well with both
catalysts. A three times excess of 2,6-dibromopyridine 1 with regard to chemical 2 was used
77
for favoring the formation of the monocoupled compound. Purification of 2 is done by
Kugelrohr followed by flash column chromatography, by which 3 is obtained with a yield of
68%. The disubstituted product 3A is still present in the purified 3, which is not a problem as
the disubstituted 3A is not reactive in the next reaction step.
Using literature conditions for the synthesis of 2-[(triisopropylsilyl)ethynyl]-6(tributylstannyl)pyridine 4, compound 9 (Figure 4) was formed almost exclusively (15). By
changing the conditions to letting the mixture stir at -78°C, both compounds 4 and 9 were
formed. The yield for the stannylated pyridine 4 was 26% after purification by column
chromatography.
Figure 4. Compounds 3A and 9, side products of the Sonogashira coupling and stannylation,
respectively.
The Stille coupling yielding the coupled product 6A was carried out with two different
catalysts, PdCl2(PPh3)2 and tetrakis. Like in the Sonogashira coupling, also for this reaction
good results were obtained with both catalysts. 4,6-bis[4’-((triisopropylsilyl)ethynyl)pyrid-2’yl]-2-phenylpyrimidine 6A was obtained with a yield of 57%. After purification by column
chromatography and recrystallization, deprotection of the alkyne functional groups was carried
out with tetra-n-butylammonium fluoride, yielding 97% protoligand 7A, which was not purified
extensively for the following Cu(I)AAC.
Synthesis azide functionalized compounds
TEG-N3. Conditions from literature were used. Trietyleneglycol mono methyl ether (TEG)
was tosylated 12 (16). After purification, TEG-tosylate 12 was reacted with sodium azide to
yield TEG-azide 13 (17).
Figure 5. Synthesis of TEG-N3
PEtOx-N3. PEtOx-N3 was prepared by CROP (cationic ring opening polymerization) of 2ethyloxazoline with MeOTs as initiator (Figure 6). The reaction was carried out in a glovebox
to ensure dry conditions and avoid termination by water. After polymerization, sodium azide is
added as terminating agent (13),(18),(19). Three different lengths were made of this azide
functionalized polymer, namely with 20, 50 and 100 repeating units. After termination the
excess of NaN3 was removed from the crude via a PD-10 desalting column.
Figure 6. Synthesis of PEtOx-N3 by CROP and termination by sodium azide
78
TABLE I. 1H NMR and DMA-SEC data
1H NMR M
Type of polymer
n
PEtOx20
2300
PEtOx50
5000
PEtOx100
8700
SEC Đ
1.08
1.07
1.06
SEC Mn
3100
7500
15500
n 1H NMR
23
50
87
n SEC
33
81
165
Figure 7. DMA-SEC (top left) and FTIR (top right) of PEtOx20, PEtOx50, PEtOx100 in black,
red and blue, respectively. MALDI-TOF (bottom left, zoom on bottom right), of PEtOx20.
The low dispersities measured with SEC indicate a well-controlled polymerization (Table
II) (18),(19). When comparing the values of Mn obtained with 1H NMR and SEC, different
values were obtained. The 1H NMR values are most reliable for Mn, as this integration is
absolute with respect to the error of integration. The deviation in Mn’s obtained by SEC is
explained by them being calculated relative to PMMA standards, which have different chemical
properties than PEtOx. The Mn’s of the different polymers were in the intended range.
Qualitative FT-IR analysis was taken (Figure 7) to visualize the azide functionality. The signal
at 1626 is the so called Amide I band, being the stretching of the C=O bond (ν (C=O)) (18),(19).
The asymmetric stretching of the azide is visible as an intense band at 2101 cm-1, which is
close to the reported wavenumbers for an azide end group on PMeOx (poly(2-methyl-2oxazoline)) at 2102 cm-1 and 2109 cm-1 (18). Moreover, while the intensity of the Amide I band
of the PEtOx20, PEtOx50 and PEtOx100 stays the same, the azide asymmetric stretching vibration
lowers in intensity. These results point out the azide functionality is attached to the polymer as
well as confirm the expected decrease in azide content with increasing polymer molar mass.
79
In the zoom of the MALDI-TOF spectrum (Figure 7, bottom right), the m/z’s of 2260, 2359
and 2458 Da correspond to the Na+ salts of PEtOx22, PEtOx23 and PEtOx24 respectively, with
99 Da the mass of the repeating units. Other recurring peaks are around 2233, 2332 and 2431
Da, which are attributed to the loss of nitrogen from the end-group (Figure 38), resulting in a
nitrene functional group (22),(23). Other distinct peaks present in the spectrum are the K+ salts.
Peaks coming from chain-transfer reactions result in a proton-initiated (13) and chain coupled
polymer (13) (Figure 8) are also present in the MALDI spectrum.
Figure 8. Side products visible in the MALDI-TOF spectrum
Cu(I)-Catalyzed Azide-Alkyne Cycloaddition
Cycloaddition of Protoligand 7A with TEG-N3 13. Synthesis of 4,6-bis[4’-((2-(2-(2methoxyethoxy)ethoxy)ethyl)-1”,2”,3”-triazol-4”-yl)pyrid-2’-yl]-2-phenylpyrimidine
8ATEG was inspired by literature conditions (24). The Cu(I) was removed by filtering over an
aluminum oxide column. Structure 8A was not obtained with these conditions and workup.
Further research is needed for carrying out this reaction step.
Figure 9. CuAAC between protoligand 7A and TEG-N3 13.
Conclusion
In this article, the synthesis of 4,6-bis[4’-(ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 7A is
discussed as well as the synthesis of TEG and PEtOx which have one azide end group. The
cycloaddition between protoligand 7A and TEG-N3 13 was not successfully carried out. Further
research is needed for finding the right conditions for this reaction. In future research, also the
cycloaddition of protoligand 7A and PEtOx-N3 will be studied, together with the
supramolecular grid formation with Fe(II) and Zn(II) of these ligands.
Acknowledgments
This work was supported by Ghent University and Supramolecular Chemistry group. A
special thanks goes to Ir. Jan Goeman and Tim Courtin for the fast analysis.
References
80
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Supporting information
Materials and equipment
Reagents were bought at Sigma Aldrich. Exceptions are TBAF 1M solution in THF, which
was bought from Fluka. 2-Ethyl-2-Oxazoline was kindly donated by Polymer Chemistry
Innovations. Proton nuclear magnetic resonance was recorded on a Bruker Avance 300 MHz at
room temperature. NMR spectra are measured in chloroform-d (CDCl3) or dimethyl sulfoxided6 DMSO-d6 (DMSO-d6) bought from Euriso-top. The chemical shifts are given in parts per
million (δ), relative to 7.26 ppm for CHCl3 and 2.54 for DMSO. Carbon 13 nuclear magnetic
resonance was recorded on a Bruker Avance II 500 MHz at room temperature. NMR spectra
are measured in chloroform-d (CDCl3) bought from Euriso-top. The chemical shifts are given
in parts per million (δ), relative to CHCl3 at 77.36 ppm.Chromatographic columns on aluminum
oxide and silica were performed on Merck Alox 90 standard aluminum oxide and on Davisil
chromatographic silica mecia LC60A 70-200 micron respectively. PD-10 columns were
performed on GE Healtcare PD-10 pre-packed columns, SephadexTM G-25M. Size-exclusion
chromatography (SEC) was performed on a Agilent 1260-series HPLC system equipped with
81
a 1260 online degasser, a 1260 ISO-pump, a 1260 automatic liquid sampler (ALS), a
thermostatted column compartment (TCC) at 50°C equipped with two PLgel 5 µm mixed-D
columns and a mixed-D guard column in series, a 1260 diode array detector (DAD) and a 1260
refractive index detector (RID). The used eluent was DMA containing 50 mM of LiCl at a flow
rate of 0.500 ml/min. The spectra were analyzed using the Agilent Chemstation software with
the GPC add on. Molar mass and PDI values were calculated against PMMA standards from
PSS. A Martin Christ Alpha 2-4 LDPlus with an ice condenser capacity of 4 kg and temperature
of -85°C and 4kg/24h performance was used as freezedryer. A Büchi GRK50 Kugelrohr was
used together with a Thyracont VD83 manometer. All FT-IR spectra were measured using
PerkinElmer Frontier FT-IR (midIR) combined with a MKII Golden Gate set-up equipped with
a diamond crystal from Specac. The spectra were measured with a wavenumber window
between 4000 and 600 cm-1. The material was placed on the measuring plate and the crystal
was brought into contact with the dry powder. LCMS analysis was performed on an Agilent
1100 HPLC with a quaternary pump and UV-DAD detection, coupled with an Agilent G1956B
MSD and an ESI ionization source. The used column was a Phenomenex - Kinetex C18 (5 µm
150x4.6 mm). A flowrate of 1.5 ml/min was used with a temperature of 35°C and an injection
volume of 15 µl. Solvent A was 5 mM NH4OAc in H2O whereas solvent B was ACN. These
solvent were used in a gradient in 6 minutes.
used, always ending in 100% of B. The mass-range was 80-1000. The positive and negative
ions are measured separately. ESI-MS analysis was performed on an Agilent 1100 HPLC with
a quaternary pump and UV-DAD detection, coupled with an Agilent G1956B MSD and an ESI
ionization source. The samples were injected by direct injection and 5 µl samples are loaded.
Solvent A was 5 mM NH4OAc in H2O whereas solvent B was ACN. A 50/50 ratio of the two
samples were used. The mass-range was 80-3000. The positive and negative ions are measured
separately. Matrix assisted laser desorption/ionization time of flight mass spectroscopy
(MALDI-TOF MS) was performed on an Applied Biosystems Voyager. The STR MALDITOF mass spectrometer equipped with 2 m linear and 3 m reflector flight tubes and a 355 nm
Blue Lion Biotech Marathon solid state laser (3.5 ns pulse). All mass spectra were obtained
with an accelerating potential of 20 kV in positive ion mode and in either reflectron or linear
mode. Trans-2-[3-(4-tert-butylphenyl)-2-methyl-2-propenylidene]malononitrile (DCTB) or 2(4’-Hydroxy-benzeneazo)benzoic acid (HABA) (20 mg/ml in acetone) were used as matrix and
NaTFA (2 mg/ml in acetone) was used as cationizing agent. Polymer samples were dissolved
in acetone (2 mg/ml). Analyte solutions were prepared by mixing 10 µl of the matrix solution
and 5 µl of the polymer solution, with or without 5 µl of the salt solution. Subsequently, 0.5 µl
of this mixture was spotted on the sample plate, and the spots were dried in air at room
temperature. A poly(ethylene oxide) standard (Mn = 2000 g/mol) was used for calibration. All
data was processed using the Data Explorer 4.0.0.0 (Applied Biosystems) software package.
Ligand Synthesis
2-Bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3. 2,6-Dibromopyridine (7759.9 mg, 32.76
mmol), PdCl2(PPh3)2 (522 mg, 0.7435 mmol) and CuI (306 mg, 1.607 mmol) were loaded in a
schlenk flask. Using a schlenk line, the schlenk flask was put under vacuum followed by argon
three times. The powders were dissolved in dry THF (70 ml) with NEt3 (8 ml), which was added
using a syringe. (Triisopropylsilyl)acetylene (2.5 ml, 11.14 mmol) was added drop wise using
a syringe and the mixture is stirred for two hours at 40°C. After the reaction, the mixture was
filtered and the solvent evaporated. The crude was loaded in a 25 ml round bottom flask by
dissolving it in DCM. This round bottom flask is put in the Kugelrohr and run at 80-90 °C by a
pressure of 1.0-0.4 mbar. The destillation is followed by TLC on silica with 85/15 nhexane/EtAc. The distillation is stopped when the spot with rf = 0.5 is gone. The content in the
82
Kugelrohr which was not distilled over was purified by flash column chromatography on silica
with n-hexane/EtAc 7/3. 2-Bromo-6-[(triisopropylsilyl)ethynyl]pyridine was obtained as a
colorless oil with a yield of 68 % (2572,6 mg, 7,6031 mmol).
1
H NMR, 300 MHz, CDCl3: 7.51-7.39 (m, 3H, pyrimidine-H), 1.13 (s, 18H, –Si-(CH(CH3)2)3), 1.12 (s, 3H, –Si-(CH-(CH3)2)3)
13
C NMR, 500 MHz, CDCl3: 143.8 (-CH=C-CΞC-TIPS); 141.6(=N-CBr=C-); 138.1 (-CHCH=C-CΞC-TIPS); 127.6 (=N-CBr=C-); 126.8 (-CH=C-CΞC-TIPS); 104.4 (=C-CΞC-TIPS-);
93.9 (=C-CΞC-TIPS); 18.6 (–Si-(CH-(CH3)2)3); 11.2(–Si-(CH-(CH3)2)3)
ESI-LCMS: [M+H]+ = 340.1
2-[(Triisopropylsilyl)ethynyl]-6-(tributylstannyl)pyridine 4. 2-Bromo-6-[(triisopropyl
silyl)ethynyl]pyridine (1010 mg, 2.988 mmol) was dissolved in THF (70 ml) and stirred under
argon atmosphere. The solution was cooled down to -78°C. Subsequently, n-BuLi (1.4 ml, 2.5
M solution in n-hexane, 3.5 mmol) was added drop wise. After stirring the mixture for 45
minutes at -78°C, a solution of Bu3SnCl (1.1 ml, 4.1 mmol) in THF (10 ml) was added drop
wise. The reaction mixture was stirred at -78°C. After six hours, the mixture was allowed to
warm to room temperature and stirred overnight. The solvents were evaporated and the residue
was purified by column chromatography on alumina with n-pentane as eluent. 2[(Triisopropylsilyl)ethynyl]-6-(tributylstannyl)pyridine was obtained as a colorless oil (476 mg,
0,868 mmol) with a yield of 29%.
1
H NMR, 300 MHz, CDCl3: 7.40 (dd, J1=6.74, J2=8.38, 1H, Bu3Sn-C-CH=CH-), 7,28 (d,
J=7,28, 1H, Sn-C-CH=CH), 7.27 (d, J=7.28, 1H, CH=C-CΞC-TIPS),1.56 (m, 6H, CH3-CH2CH2-CH2-Sn-), 1.33 (s, J=7.5Hz, 6H, CH3-CH2-CH2-CH2-Sn-), 1.15 (s, 18H, –Si-(CH(CH3)2)3), 1.14 (s, 3H, –Si-(CH-(CH3)2)3), 1.10 (m, 6H, CH3-CH2-CH2-CH2-Sn-), 0.88 (t,
J=7.27, 9H, CH3-CH2-CH2-CH2-Sn-)
13
C NMR, 500 MHz, CDCl3: 174.6 (Bu3Sn-C=C-); 144.1 (-CH=C-CΞC-TIPS); 132.6
(Bu3Sn-C=C-C=); 130.9 (Bu3Sn-C=C-); 126.1 (-CH=C-CΞC-TIPS); 107.4 (=C-CΞC-TIPS);
90.5 (=C-CΞC-TIPS); 29.0 (CH3-CH2-CH2-CH2-Sn-); 27.0 (CH3-CH2-CH2-CH2-Sn-); 18.7 (–
Si-(CH-(CH3)2)3); 13.7 (–Si-(CH-(CH3)2)3); 11.4 (CH3-CH2-CH2-CH2-Sn-); 10.1 (CH3-CH2CH2-CH2-Sn-)
ESI-LCMS: [M+H]+ = 260.1
4,6-Bis[4’-((triisopropylsilyl)ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 6A. 2-[(Triiso
propylsilyl)ethynyl]-6-(tributylstannyl)pyridine 4 (43 mg, 0.078 mmol), fenclorim 5A (8.2 mg,
36 mmol) and PdCl2(PPh3)2 (2.8 mg, 3.9 µmol) were brought in a dry round bottom flask and
3.5 ml of dry DMF was added. The mixture was stirred under an Ar atmosphere at 80°C for 42
hours. The solvent was evaporated and a brown oil was obtained, which was purified by column
chromatography on silica with n-pentane/DCM (starting at 100/0 to 70/30) with a drop of NEt3.
The silica was first purged with n-pentane and a drop of NEt3. The column is followed by TLC
with n-pentane/DCM 85/15 and a drop of NEt3 to determine which fraction contains the clean
product. This fraction is then recrystallized in with EtOH, starting from EtOH under reflux and
letting it slowly cool down to get the white crystalline 4,6-bis[4’((triisopropylsilyl)ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 6A with a yield of 57% (14 mg,
0.021 mmol). The reaction was also carried out with Pd(PPh3)4 (4.5 mg, 3.9 µmol) following
the same procedure.
1
H NMR, 300 MHz, CDCl3: 9.30 (s, 1H, -N=C(-pyridine)-CH=C(-pyridine)-N=); 8.72-8.69
(m, 2H, =CH-C(-pyrimidine)=CH-); 8.66 (dd, J1=1.05, J2=7.93, 2H, -N=(pyrimidine-)CCH=CH- CH=C-CΞC-TIPS); 7.85 (t, J=7.82, 2H, -N=(pyrimidine-)C-CH=CH-CH=C-CΞCTIPS); 7.61 (dd, J1=1.06, J2=7.70, 2H, -N=(pyrimidine-)C-CH=CH-CH=C-CΞC-TIPS); 7.5983
7.52 (m, 3H, -CH=CH-CH=CH-C(-pyrimidine)=CH-); 1.20 (s, 36H, –Si-(CH-(CH3)2)3); 1.19
(s, 6H, –Si-(CH-(CH3)2)3)
ESI-MS: [M+H]+= 671.4
4,6-Bis[4’-(ethynyl)pyrid-2’-yl]-2-phenylpyrimidine
7A.
4,6-Bis[4’-((triisopropyl
silyl)ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 6A (27 mg, 0.0402 mmol) was dissolved in THF
(4 ml) in a round bottom flask and stirred. TBAF (1M solution in THF, 0.15 ml, 0.15 mmol)
was added dropwise. The solvent was evaporated after which the product was dissolved in
water. Chloroform was added and the chloroform phase was extracted 3 times more with water.
A brown oil was obtained after evaporation of the chloroform. The oil is purified by a column
chromatography on silica with n-pentane/DCM/MeOH in a gradient starting from 85/15/0 to
0/90/10 with 0.5% of NEt3. Afterwards, the product was dissolved in CHCl3 and washed with
water to get rid of the NEt3. 4,6-Bis[4’-(ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 7A was
obtained as a yellow solid with a yield of 97% ( 14 mg, 0,038 mmol).
1
H NMR, 300 MHz, CDCl3: 9.29 (s, 1H, -N=C(-pyridine)-CH=C(-pyridine)-N=); 8.728.69 (m, 4H, =CH-C(-pyrimidine)=CH-, -N=(pyrimidine-)C-CH=CH-CH=C-CΞC-TIPS); 7.89
(t, J=7.83, 2H, -N=(pyrimidine-)C-CH=CH-CH=C-CΞC-TIPS); 7.62 (dd, J1=1.06, J2=7.70, 2H,
-N=(pyrimidine-)C-CH=CH-CH=C-CΞC-TIPS); 7.59-7.51 (m, 3H, -CH=CH-CH=CH-C(pyrimidine)=CH-); 3.25 (s, 2H, H-CΞC-)
ESI-MS: [M+H]+ = 359.2
Polymer and Oligomer Synthesis
2-(2-(2-Methoxyethoxy)ethoxy)ethyl tosylate 12. Triethylene glycol monomethyl ether
(9.7 ml, 61 mmol), NEt3 (21.2 ml, 0,1525 mol) and 4-dimethylaminopyridine, DMAP (1.74 g,
14.2 mmol) were dissolved in DCM (400 ml). The mixture was cooled to 0°C and 4toluenesulfonylchloride (13.2 g, 69 mmol) was added. After several minutes, the solution got
an orange color and the ice bath was removed. The mixture was stirred overnight at room
temperature. The solution was washed with water and dried over MgSO4. After evaporation of
the solvent, column chromatography was used to purify the compound with silica as stationary
phase and ethyl acetate/DCM 3/7 as the eluent. 2-(2-(2-Methoxyethoxy)ethoxy)ethyl tosylate
was obtained as a yellow oil (13,45 g, 42.3 mmol, 70 %).
1
H NMR, 300 MHz, CDCl3: 7.77 (d, J=8.3 Hz, 2H, RO3S-C-CH=CH-), 7.33 (m, J=8.0, 2H,
RO3S-C-CH=CH-), 4.41 (m,2H, Tos-CH2-), 3.65 (m, 2H, Tos-CH2-CH2-O-), 3.56 (m, 6H, CH3O-CH2-CH2-O-CH2-) 3.51 (m, 2H), 3.34 (s, 3H, CH3-O-CH2-), 2.41 (s, 3H, RO3S-C-CH=CHC-CH3)
ESI-LCMS (conditions): [M+NH4]+=336.2
2-(2-(2-Methoxyethoxy)ethoxy)ethyl azide 13. 2-(2-(2-Methoxyethoxy)ethoxy)ethyl
tosylate (3 g, 0.18 mol) was dissolved in 20 ml of purified DMF under argon. NaN3 (2.11 g,
0.62 mol) was slowly added with a plastic spoon. The reaction was stirred at 60°C. After 48
hours, 60 ml water was added to the mixture. An extraction was performed with diethylether.
The organic phase was dried with MgSO4, filtered and dried under vacuum. A yellow oil was
obtained. The solvent was not completely evaporated for a more save storage.
1
H NMR, 300 MHz, CDCl3: 3.64 (m, 8H, CH3-O-CH2-CH2-O-CH2- CH2-O-), 3.52 (m, 2H,
-O-CH2-CH2-N3), 3.36 (m, 5H, CH3-O-CH2-CH2-O-CH2- CH2-O-CH2-CH2-N3)
PEtOx20-N3. EtOx and MeOTs were put in a round bottom flask in a glove box, dissolved
in ACN (1.789 ml) and stirred at 100°C. After the reaction time given in Table II, NaN3 was
added. The mixture was taken out of the glovebox and the solvent was evaporated. Water was
84
added and the solution was purified on a PD-10 desalting column. After freeze drying, 0.3984
mg of PEtOx20-N3 was obtained. Synthesis of PEtOx50-N3 and PEtOx100-N3 was performed
using similar conditions and details are shown in Table II.
1
H NMR, 300 MHz, CDCl3: 0.96-1.20 (300 H, CH3-[N((C=O)-CH2-CH3)-CH2-CH2]-N3);
2.25-2.50 (200H, CH3-[N((C=O)-CH2-CH3)-CH2-CH2]-N3); 3.00 (PEtOx20:13.2 H; PEtOx50:
6.0H; PEtOx100: 3.3H, CH3-[N((C=O)-CH2-CH3)-CH2-CH2]-N3); 3.43 (400 H, CH3-[N((C=O)CH2-CH3)-CH2-CH2]-N3)
TABLE II. Masses, volumes, molarities, reaction times and yields of the PEtOx-N3
Type of polymer
EtOx
MeOTs
NaN3
PEtOx20
1.211 ml; 12.0 mmol
90.84 µl; 0.652 mmol
177 mg; 1.80 mmol
PEtOx50
1.211 ml; 12.0 mmol
36.3 µl; 0.241 mmol
47 mg; 0.73 mmol
PEtOx100
1.211 ml; 12.0 mmol
8.6 µl; 0.057 mmol
23 mg; 0.36 mmol
t reaction (min)
25
65
120
Yield
0.398 mg; 40%
0.615; 52%
0.786; 66%
Cu(I)-Catalyzed Azide-Alkyne Cycloadditions
4,6-Bis[4’-((2-(2-(2-Methoxyethoxy)ethoxy)ethyl)-1”,2”,3”-triazol-4”-yl)pyrid-2’-yl]-2phenylpyrimidine 8A-TEG. 4,6-Bis[4’-(ethynyl)pyrid-2’-yl]-2-phenylpyrimidine 7A (13.7 mg,
38.2 µmol), 2-(2-(2-methoxyethoxy)ethoxy)ethyl azide 13 (95.2 mg; 502 µmol) and Na
ascorbate (1.87 mg, 9.44 µmol) were brought in a microwave vial. A solution of CuBr (12.0
mg, 83.7 µmol) and PMDETA (14.5 mg, 83.7 µmol) in dry DMF (12 ml) was added. The
mixture was stirred overnight under Ar at room temperature. The next day, the mixture has
turned deep purple. THF (12 ml) was added and the mixture was filtered over aluminum oxide
with THF as eluent. Subsequently, DCM was used remove everything from the aluminum
oxide. A green band was visible at the top of the aluminum oxide, indicating successful removal
of the copper. The copper was removed from the column when eluting with 20% MeOH and a
few drops of NEt3. Afterwards, the solvent was evaporated. 6-Bis[4’-((2-(2-(2methoxyethoxy)ethoxy)ethyl)-1”,2”,3”-triazol-4”-yl)pyrid-2’-yl]-2-phenylpyrimidine
8ATEG was not obtained.
85
86
7 Dutch Summary
Een nieuw ligand met als doel het vormen van een supramoleculaire grid structuur werd
ontworpen. Supramoleculaire interacties zijn de interacties die voorbij de chemisch covalente
binding liggen, zoals waterstof bruggen, π-π interacties en, de belangrijkste met oog op het
grid, de metal-ligand wisselwerking. Een supramoleculaire grid structuur is de structuur
gevormd door vier liganden en vier metaalionen, waarbij een de vorm van deze
supramoleculaire eenheid de vormen van een rooster aanneemt. Het hier ontwikkelde ligand
is ontworpen om complexen te vormen met octaëdrisch coördinerende metaalionen, zoals
Zn(II) en Fe(II). Met tetraëdrisch coördinerende metal kationen, zoals Cu(I), wordt niet het grid
gevormd, maar kan een helix vormig complex met twee Cu(I) ionen en drie liganden ontstaan.
Reden voor de keuze van deze octaëdrische omwikkeling is dat de metaal ionen in water
gebruikt kunnen worden. Het hier ontwikkelde ligand bevat twee triazole ringen. Deze worden
door koper(I) gekatalyseerd azide-alkyn cycloadditie (CuAAC) gevormd, waardoor oligo- en
polymeren aan het grid kunnen gehangen worden. Het uiteindelijke doel van het ligand is om
het te incorporeren in een hydrogel waarin het als een supramoleculaire crosslinker kan
dienen. Dit met de intentie om de hydrogel zelfhelende en afschuiving verdunnende
macromoleculaire eigenschappen te geven.
De syntheseroute van het ligand 8 is te vinden in Figuur 16. De eerste reactie stap is een
Sonogashira koppelingsreactie tussen triisopropylsilyl-acetyleen 1 en 2,6-dibromopyridine 2,
gekatalyseerd door Pd katalysatoren. De katalysatoren zijn zuurstof gevoelig en zuurstofvrij
werken is een vereiste. Twee verschillende Pd katalysatoren werden gebruikt en met beide
werd een bevredigend resultaat bekomen. Het opzuiveren van 3 met een chromatografische
kolom was geen sinecure, gezien de weinige scheiding in termen van rf waarden. Een
Kugelrohr in combinatie met flash-chromatografie loste deze complicatie op.
De stannylatie van 3 gebeurde door eerst n-BuLi toe te voegen, gevolgd door de toevoeging
van Bu3SnCl, waardoor het broom atoom gesubstitueerd wordt door een Bu3Sn functionele
groep. Literatuur condities voor synthese van deze stof waren niet ideaal, gezien het door
waterstof gesubstitueerde product hier voornamelijk gevormd werd, en het gestannyleerde
pyridine niet. Het zuiverder maken van de beginproducten loste dit knelpunt niet op.
87
Uiteindelijk werden de condities gevarieerd waardoor het juiste gestannyleerde product 4
bekomen kon worden.
Vervolgens werd een Stille koppeling met 4,6-diiodopyrimidine 5 geprobeerd. Gezien het niet
werken van deze reactie werd opnieuw de zuiverheid van de componenten nagekeken en
twijfeling over zuiverheid van 4,6-diiodopyrimidine 5 maakte dat eerst andere routes gevolgd
werden. Geopteerd werd om 4,6-dichloro-2-phenylpyrimidine 5A (fenclorim) te gebruiken, dit
in plaats van het diiodopyrimidine 5. Met fenclorim in plaats van stof 5 kon de
koppelingsreactie wel uitgevoerd worden (Figuur 25) en werd 6A bekomen. Dit fenclorim
heeft nog een fenylgroep dewelke 5 niet heeft. De aanwezigheid van die fenylgroep is
functioneel voor de supramoleculaire grid structuur, aangezien deze fenylgroep daar in het
midden kan komen en zo, via π-π stapeling een extra stabiliserende kracht kan geven. Ook
voor deze reactie werden twee verschillende katalysatoren gebruikt die beide geschikt waren.
Na opzuiveren werd ontscherming van de TIPS beschermingsgroep uitgevoerd met TBAF.
Protoligand 7A werd bekomen.
De oligomeren en polymeren die geklikt werden zijn TEG (triethylenglycol monomethyl ether)
13 en PEtOx (Poly-(2-ethyl-2-oxazoline)), beide met azide eind groep functionaliteit. De
synthese van TEG werd uitgevoerd door het vrije alcohol van TEG the tosyleren. Nadien werd
een substitutie reactie met natrium azide uitgevoerd. PEtOx werd door kationische
ringopeningspolymerisatie uitgevoerd. Natrium azide werd hier als terminator aan
toegevoegd voor de azide functionaliteit in te voeren. De polymeren werden gekarakteriseerd
door 1H NMR en DMA-SEC, waardoor de Mn en dispersiteit mee kon bekomen worden. Met
FTIR en MALDI-TOF werd de azide eindgroepsfunctionaliteit aangetoond.
De koper(I) gekatalyseerde cycloadditie tussen TEG-azide 13 en protoligand 7A werd
bestudeerd, resulterend in het ligand 8A-TEG (Figuur 39), maar deze reactie is nog niet
succesvol uitgevoerd.
De cycloadditie tussen PEtOx en protoligand 7A, alsmede of deze liganden wel degelijk
supramoleculaire grid structuren zijn onderwerpen voor verder onderzoek. Ook is de
bestemming van de integratie van deze supramoleculaire grids als crosslinker in hydrogelen
nog niet bereikt, een bijkomend argument voor vervolgende studies omtrent dit werk.
88
8 Supporting Info
SI 1: 1H NMR spectrum of 4,6-diiodopyrimidine. At 7,26 is the CHCl3 peak visible.
T (°C)
70
80
85
90
95
100
110
120
255
P (mbar)
0,66
1,22
1,73
2,42
3,36
4,44
7,75
12,26
1000,00
SI 2: Boiling points of 2,6-dibromopyridine 1 at different pressures.
89
SI 3: 1H NMR spectrum of purified 2-[(triisopropylsilyl)ethynyl]pyridine 9
SI 4: 1H NMR spectrum of purified 2-[(triisopropylsilyl)ethynyl]-6-(tributylstannyl)pyridine 4
90
SI 5: 1H NMR spectrum of 2-bromo-6-[(triisopropylsilyl)ethynyl]pyridine 3 purified by
Kugelrohr
SI 6: 1H NMR spectra of PEtOx20-N3, PEtOx20-N3, PEtOx50-N3 and PEtOx100-N3 measured in
DMSO-d6
91
SI 7: PEtOxN3: In grey is the 1H NMR spectrum before adding water, in black after. The peak at
2,15 shifted while a peak a 4,75 arose.
SI 8: 1H NMR spectra of PEtOx20 -N3, PEtOx50 -N3 and PEtOx100 -N3 in CDCl3
92
SI 9: 1H NMR spectra of 2-(2-(2-methoxyethoxy)ethoxy)ethyl tosylate 12 (top) and 2-(2-(2methoxyethoxy)ethoxy)ethyl azide 13 (bottom).
93
600
400
400
Intensity
Intensity
600
200
200
0
0
4000
5000
5050
5100
200
200
150
150
100
5200
5250
5300
100
50
50
0
0
8000
5150
Mass (m/z)
Intensity
Intensity
Mass (m/z)
9150
10000
9200
9250
9300
9350
9400
Mass (m/z)
Mass (m/z)
SI 10: MALDI-TOF results of PEtOX50 (top) and PEtOx100 (bottom). Left the broad spectrum,
right a zoom.
SI 11: LCMS analysis of 4,6-diiodopyrimidine (which has an exact molar mass of 331,8). Top:
UV detection. Middle: Total ion count. Bottom: Extracted ion chromatogram of m/z 332 and
333.
94
SI 12: Mass spectrum of SI 11 at a retention time of 5,707. [M+H]+ = 332,6 is visible. A lot of
other impurities are also visible.
95
SI 13: 1H NMR spectra of an old bottle of Bu3SnCl (bottom) and a new bottle of Bu3SnCl (top).
The peaks underlined in black are proposed as Sn-H satellites, coming from the coupling of
117Sn and 119Sn, which have J-coupling constants in the order of 20-30 Hz for J and J couplings
2
3
82
as is the case here . Also the relative integration of the proposed satellite peaks are in
correspondence for them being satellites, as the 117Sn and 119Sn are the only NMR active nuclei
which have a reasonable abundancy with a relative abundancy of 7.7 % and 8.6 % respectively.
Other indication which made the suspicion of impurities in the Bu3SnCl were the small peaks
under the big peaks of protons Ha. As both the 1H NMR spectra of the old bottle of Bu3SnCl
and of the new bottle looked the same, these peaks are also not attributed to impurities. The
small peaks between the peaks of protons Ha might come from conformational differences
between different Bu3SnCl molecules. If the underlined peaks and the small peaks under
hydrogens Ha would however be impurities, as the compound is 96% clean according to SigmaAldrich, further reactions with this Bu3SnCl proved to be successful, indicating possible
impurities in the Bu3SnCl were not to blame for failure of the reaction at this stage.
96