GHENT UNIVERSITY
FACULTY OF SCIENCES
Department of Organic Chemistry Supramolecular Chemistry Group
Academic year 2011-2012
Florian BONTE
First master of drug development
Promotors Dr. Ir. B. De Geest
Prof. Dr. Ir. R. Hoogenboom
Commissioners Dr. Ir. B. De Geest
Prof. Dr. Ir. R. Hoogenboom Prof. Dr. T. De Beer
Evaluation of poly(2-oxazoline) derivatives as poly(ethylene glycol) alternatives
GHENT UNIVERSITY
FACULTY OF SCIENCES
Department of Organic Chemistry Supramolecular Chemistry Group
Academic year 2011-2012
Florian BONTE
First master of drug development
Promotors Dr. Ir. B. De Geest
Prof. Dr. Ir. R. Hoogenboom
Commissioners Dr. Ir. B. De Geest
Prof. Dr. Ir. R. Hoogenboom Prof. Dr. T. De Beer
Evaluation of poly(2-oxazoline) derivatives as poly(ethylene glycol) alternatives
CONFIDENTIALITY AGREEMENT
The reader of this work is required to respect the confidentiality of the contents disclosed
herein. Reproduction of the entire text or parts thereof is prohibited. You are kindly asked to
return the copy of this work that was handed out to you back to the promoter, Dr. Ir. B. De
Geest.
The promoters
The commissioner
Name: Dr. Ir. B. De Geest Name: Prof. Dr. T. De Beer Address: Laboratory of Pharmaceutical Technology Harelbekestraat 72 9000 Ghent
Adress: Laboratory of Pharmaceutical Process Analytical Technology Harelbekestraat 72 9000 Ghent
Date and signature
Date and signature
Name: Prof. Dr. Ir. R. Hoogenboom Adress: Supramolecular Chemistry Group Krijgslaan 281 S4 9000 Ghent
Date and signature
SUMMARY
Poly(ethylene glycol) (PEG) is a well-established stealth polymer, used for already more
than 20 years to enhance blood circulation times of drugs (mostly biomolecules or
liposomes). This enhanced circulation time is due to the increased hydrodynamic radius,
which is related to the molecular weight preventing excretion by the kidneys. The increased
water solubility and highly hydrated structure of PEG prevents metabolism and recognition
by the immune system.
Some disadvantages of PEG have also been reported. Immunological responses,
accelerated clearance and accumulation of PEG are the most important disadvantages. Due
to these drawbacks different polymers are being investigated as alternative stealth
polymers. Amongst the most important alternatives are the poly(cyclo imino ether)s. The
polymer architecture can be varied (both side chain and main chain) to induce changing
properties like thermosensitivity and water solubility. The most common poly(cyclic imino
ethers) investigated are poly(oxazoline)s but insertion of a methylene group into the main
chain yields the poly(oxazine)s, which have been much less studied.
Poly(2-methyl-2-oxazine) (PMeOz) is proposed to be a good alternative as stealth
polymer to PEG, because the water solubility lies theoretically between that of poly(2-
methyl-2-oxazoline) (PMeOx) (which is too water soluble to conjugate to a biomolecule in
organic solvents) and that of poly(2-ethyl-2-oxazoline) (PEtOx) (which has a water solubility
comparable to PEG).
To confirm this hypothesis, PMeOx and PEtOx were synthesized as reference polymers
as well as PMeOz. Kinetic studies of the synthesis of PMeOx, PEtOx and PMeOz by cationic
ring-opening polymerization confirmed the livingness of the polymerisation reaction. Based
on the kinetic studies, polymers with different degree of polymerization were prepared.
To synthesise the polymers, monomers and initiator were needed in pure form which
was obtained by distillation. In addition the 2-methyl-2-oxazine was not commercially
available, and had to be synthesised.
Then the hydrophilicity of PMeOx, PEtOx and PMeOz were measured with RP-HPLC.
The results show that the water solubility of PMeOz indeed lies in between the two
reference polymers, but nearer to PMeOx than to PEtOx.
SAMENVATTING
Poly(ethyleen glycol) (PEG) is een stealth polymeer dat al meer dan 20 jaar op de
markt is om de retentietijd van geneesmiddelen (meestal biomoleculen of liposomen) in het
lichaam te verlengen. Dit is gerelateerd aan een vergroting van de hydrodynamische radius,
die afhangt van het moleculair gewicht. Dit verhindert excretie door de nieren. De
verhoogde wateroplosbaarheid vermindert de metabolisatie en de herkenning door het
immuunsysteem.
Er zijn echter ook nadelen gerapporteerd over PEG. Immunologische reacties,
versnelde klaring uit het bloed en opstapeling van PEG zijn de belangrijkste problemen.
Vanwege deze nadelen werden en worden verschillende polymeren onderzocht als
alternatieve stealth polymeren. Waarschijnlijk het belangrijkste alternatief is de groep van
de poly(cyclo imino ether)s. De opbouw van het polymeer kan gevarieerd worden zowel in
de zijketen als in de hoofdketen, wat leidt tot veranderingen in eigenschappen zoals
thermoresponsiviteit en wateroplosbaarheid. De best onderzochte poly(cyclo imino ether)s
zijn de poly(oxazoline)s, maar door het invoegen van een methyleen groep in de hoofdketen,
verkrijgt men de poly(oxazine)s.
Poly(2-methyl-2oxazine) (PMeOz) is een veelbelovend alternatief als stealth polymeer
voor PEG, omdat de wateroplosbaarheid waarschijnlijk ligt tussen die van poly(2-methyl-2-
oxazoline) (PMeOx) (die te wateroplosbaar is om te koppelen aan een biomolecule) en
poly(2-ethyl-2-oxazoline) (PEtOx) (dat een wateroplosbaarheid heeft vergelijkbaar met die
van PEG).
Om deze hypothese te bevestigen, werden PMeOx en PEtOx gesynthetiseerd als
referentiepolymeer, evenals PMeOz. Kinetische studies werden uitgevoerd van de synthese
van PMeOx, PEtOx en PMeOz via de kationische ring-openingpolymerisatiereactie. Deze
toonden aan dat de polymerisatie levend is. Gebaseerd op deze kinetische studies werden
polymeren met een verschillende polymerisatiegraad aangemaakt.
Voor de synthese van de polymeren waren zuivere initiator en monomeren nodig,
deze werden verkregen na destillatie. Enkel de 2-methyl-2-oxazine was niet commercieel
beschikbaar en moest gesynthetiseerd worden.
Daarna werd de hydrofiliciteit van PMeOx, PEtOx en PMeOz gemeten met RP-HPLC. De
resultaten suggereren dat de wateroplosbaarheid van PMeOz inderdaad tussen de twee
referentiepolymeren ligt, maar dichter bij de PMeOx dan bij de PEtOx.
ACKNOWLEDGMENTS
Firstly, I want to thank Victor R. De La Rosa, my mentor for this thesis. With a lot of patience
he explained me what I had to do, and he learned me a lot of things.
I also want to thank Prof. Richard Hoogenboom for helping me if I had a question or a
problem and pushing me in the right direction for my thesis.
Thanks to Bruno De Geest for giving me this place for my thesis in a real chemistry lab.
I also want to thank Daniel Frank, because he helped me a lot with my practical work by
answering my questions and explaining how to carry out certain experiments.
A lot of thanks to Jos Van den Begin, because he helped me with my HPLC-experiments.
Furthermore, I want to thank Bryn Monnery, for synthesizing the methyloxazine, so I could do
the kinetic experiments of the polymerization of methyloxazine.
Thanks also to Alessandro Tavecchia, for the coffee that allowed me to do my work properly.
And last but not least I want to thank the rest of the people of the lab for helping me,
explaining me, answering me, being kind: Gertjan Vancoillie, Maarten Mees, Lenny
Voorhaar, Qilu Zhang, Bram Denhaerinck, Kanykei Ryskulova and Maji Samarendra.
TABLE OF CONTENTS
1. INTRODUCTION ..................................................................................................................................... 1
2. OBJECTIVES .......................................................................................................................................... 11
3. MATERIALS AND METHODS ................................................................................................................. 13
3.1. MATERIALS .............................................................................................................................................. 13
3.1.1. Monomers ................................................................................................................................ 13
3.1.2. Chemicals for the synthesis of the 2-methyl-2-oxazine monomer ............................................ 13
3.1.3. Chemicals for the polymer synthesis ........................................................................................ 13
3.1.4. Chemicals for evaluation of the watersolubility-related properties ......................................... 13
3.1.5. General solvents and salts ........................................................................................................ 14
3.1.6. Devices ...................................................................................................................................... 14
3.2. METHODS ................................................................................................................................................ 15
3.2.1. Distillation ................................................................................................................................ 15
3.2.2. Synthesis of the 2-methyl-2-oxazine ......................................................................................... 16
3.2.3. Polymerization of 2-oxazolines ................................................................................................. 17
3.2.4. Kinetic studies ........................................................................................................................... 18
3.2.4.1. Polymerization kinetics of poly(2-methyl-2-oxazoline) ............................................................................. 18
3.2.4.2. Polymerization kinetics of poly(2-ethyl-2-oxazoline) ................................................................................ 19
3.2.4.3. Polymerization kinetics of poly(2-methyl-2-oxazine) ................................................................................ 19
4. RESULTS AND DISCUSSION .................................................................................................................. 20
4.1. PURIFICATION OF MEOTS, MEOX AND ETOX ............................................................................................... 20
4.2. SYNTHESIS OF THE 2-METHYL-2-OXAZINE ............................................................................................ 20
4.2.1. Cyclo condensation of 3-amino-1-propanol and acetonitrile ................................................... 20
4.2.2. Synthesis of MeOz via the acid amide ...................................................................................... 22
4.3. POLYMERIZATION: GENERAL ASPECTS ............................................................................................................. 24
4.3.1. Reaction mechanism ................................................................................................................ 25
4.3.2. Determination of the monomer conversion ............................................................................. 26
4.3.3. Characterization ....................................................................................................................... 26
4.4. KINETIC STUDIES ........................................................................................................................................ 27
4.4.1. Kinetic studies of PMeOx .......................................................................................................... 29
4.4.2. Kinetic studies of PEtOx ............................................................................................................ 31
4.4.3. Kinetic studies of PMeOx .......................................................................................................... 32
4.5. POLYMER SYNTHESIS .................................................................................................................................. 33
4.5.1. Precipitation and characterization ........................................................................................... 33
4.5.2. 1H NMR spectra ........................................................................................................................ 33
4.6. WATER SOLUBILITY RELATED TESTS ON THE POLYMERS ...................................................................................... 37
4.6.1. Viscosimetry ............................................................................................................................. 37
4.6.2. RP-HPLC measurements of the tested polymers ...................................................................... 37
5. CONCLUSIONS ..................................................................................................................................... 43
6. REFERENCES ........................................................................................................................................ 43
ABBREVIATIONS:
ACN: acetonitrile
API: active pharmaceutical ingredient
DCM: dichloromethane
DMA: N,N-dimethylacetamide
DP: degree of polymerization
GC: gas chromatography
1H NMR: proton nuclear magnetic resonance (spectroscopy)
M/I: monomer/initiator-concentration ratio
M/I100: polymer with a chain length of 100 repeating units.
M/I200: polymer with a chain length of 200 repeating units.
M/I50: polymer with a chain length of 50 repeating units.
MeOTs: methyl-p-toluenesulfonate (methyl tosylate)
Mn: number-average molecular weight
PDI: polydispersity index
PEG: poly(ethylene glycol)
PEtOx: poly(2-ethyl-2-oxazoline)
PEtOz: poly(2-methyl-2-oxazine)
PMeOx: poly(2-methyl-2-oxazoline)
PMeOz: poly(2-methyl-2-oxazine)
POx(s): poly(oxazoline)(s)
POz(s): poly(oxazine)(s)
PPrOx: poly(2-n-propyl-2-oxazoline)
PPrOz: poly(2-n-propyl-2-oxazine)
RI: refractive index
RP-HPLC: reversed phase- high performance liquid chromatography
SEC: size exclusion chromatography
THF: tetrahydrofuran
1
1. INTRODUCTION
PEG (Poly(ethylene glycol, general structure is depicted
in Fig.1.1) is a polymer that is used in pharmaceutical
formulations to conjugate with drugs, biomolecules, liposomes
or micelles, to prolong the residence time in the body and
increase solubility and biocompatibility. The first research about PEG was done in 1970 by
Davis, Abuchowski and co-workers. They foresaw the potential of conjugating PEG to
proteins.1,2,3 This technique is now called ‘PEGylation’. The first PEGylated products were
marketed 20 years ago. The properties that are given to the drug, biomolecule, liposome or
micelle (hereafter called ‘the drug’ or ‘active pharmaceutical ingredient, API’) due to
PEGylation can be summarized as ‘stealth behaviour’. This means that the conjugate will stay
longer in the body than the non-PEGylated API, while the API will be protected from
biological degradation and excretion. This has several implications that are discussed
hereafter.4
Firstly, conjugating a drug with PEG will increase the molecular weight, and this in turn
limits the excretion by the kidney. The fenestrae in the blood capillaries of the kidney are too
small, which prevents the conjugates from leaving the blood stream and moving to the
urine.
A second cause for the prolonged residence in the blood circulation is that PEG is a
hydrophilic molecule. This results in a higher water solubility of the drug and an inhibition of
the opsonisation. Because the highly hydrated PEG-structure resembles the water structure,
the drug is not recognized by the immune system (due to stealth behaviour).
A third property of PEG that leads to a higher blood circulation time is steric hindrance.
The drug is sterically protected by the PEG chains against degradation by enzymes (for
example, proteases in the case of proteins).
A PEGylated product also shows a lower receptor-mediated uptake by the cells of the
organs of the reticuloendothelial system (system of monocytes and macrophages in inter
alia liver and spleen), resulting in a decreased metabolisation rate.
FIGURE 1.1: Structure of PEG
2
All these properties contribute to a longer circulation time in the blood stream, and,
thus an enhanced drug activity, because the drug to which PEG is attached has more chances
to interact with its target. In consequence, less of the drug has to be taken to achieve the
same effect, which results in lower costs and decreased undesired side effects. The drug can
also be administrated in a less frequent dosage, which results in a better adherence of the
patient to the drug.5,6
Another good property of PEG-conjugates is that PEG sterically prevents the
aggregation of the drugs during storage, which leads to higher stability and, thus, to a later
expiration date.
PEG conjugates and PEGylated carriers can also be used for passive targeting in tissues
by the enhanced permeation and retention effect (EPR-effect).7 This effect is seen in cancer
and inflamed tissue, and is caused by hypervascularisation and porous blood vessels. Large
molecules can more easily enter the inflamed or cancerous tissue through the large pores.
The molecules remain inside, because there is also an enhanced retention effect due to a
lower lymphatic drainage. In this way, tissues with leaky vasculature are preferentially
entered, which is called ‘passive targeting’.7,8,9
Unfortunately, PEG has also some disadvantages.4 Even though it prevents recognition
by the immune system, immunological responses have been reported against PEG after
intravenous administration, with blood clotting as a result.10
A second drawback is that, in some cases, an accelerated clearance from the blood was seen
of the conjugate after second injection.11 Another problem is that the human body cannot
degrade PEG. If the conjugate is not excreted by the kidneys, there is a risk of accumulation
of the conjugate, which can result in toxicological side-effects.
Because of these undesirable effects of PEG, there is an enhanced interest for
alternatives.4,12 Different synthetic polymers were investigated:
- Poly(amino acids): biocompatible and easily biodegradable
- Poly(glycerol): similar to PEG
- Vinyl polymers (poly(acrylamide), poly(vinylpyrrolidone), poly(N-(2-
hydroxypropyl)methacrylamide)
3
- Poly(oxazoline)s (POx) and poly(oxazine)s (POz) (General structures are depicted in
Fig.1.2)
The last group of alternatives contains polymers with positive properties with respect to
stealth behaviour. By analogy to ‘PEGylation’, conjugating a molecule with poly(2-oxazoline)s
or poly(2-oxazine)s is regarded as ‘POXylation’. The research described in this master’s thesis
is about this class of polymers.
FIGURE 1.2: Left: General structure of the poly(2-oxazoline)s. R=Me: PMeOx; R=Et: PEtOx Right: General structure of the poly(2-oxazine)s. R=Me: PMeOz; R=Et: PEtOz
Poly(2-oxazoline)s (more specifically the poly(2-methyl-2-oxazoline) and the poly(2-
ethyl-2-oxazoline)), were discovered in the 1960s. In that period, the polymerization by
cationic ring opening was discovered independently by four research groups.13,14,15,16
However, the general awareness about the possible biomedical applications arose only
recently, although the concept of POXylation was first suggested 20 years ago.
POxs are not only used because of their stealth behaviour, but can be used and are currently
investigated for various different applications.17
Firstly, they have a lot of biomedical applications. One of those result (as already
mentioned) from the stealth behaviour: coupled to for example biomolecules, micelles,
liposomes and drugs, it enhances the residence time and biocompatibility of the drug in the
body.
To use poly(2-oxazoline)s for this purpose, they must be compatible to the body. This was
investigated by Goddard et al. via intravenous injection in mice.18 The polymers were
excreted without significant accumulation in organs, exhibiting biocompatibility properties
matching those of the gold standard PEG.
A first proof for the stealth behaviour of poly(2-oxazoline)s was given by Dejardin et al.
They showed that a poly(2-oxazoline) triblock copolymer suppressed platelet inhibition and
fibrinogen adsorption.19 The ultimate proof for the biocompatibility and stealth behaviour of
4
poly(2-methyl-2-oxazoline) (PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx), conjugated to
liposomes, was given by Zalipsky et al.20,21 Enhanced circulation times were found for
liposomes coated with PMeOx, PEtOx and PEG, with similar blood clearance rates for PMeOx
and PEG, while PEtOx was eliminated even faster. They also showed that the three polymers
had a similar distribution profile in organs (liver, spleen and kidney) after 24h.
Biodistribution and excretion of radiolabeled poly(2-alkyl-2-oxazoline)s in mice was
studied by Jordan, Essler and co-workers.22 They found that the polymers did not
accumulate in tissues and were rapidly cleared from the blood stream, mostly by glomerular
filtration in the kidneys. Fig. 1.3, adopted from reference 22, shows the distribution profile
of the polymers.
Chung and co-workers investigated blood compatibility of PEtOx in vitro. PEtOx
suppressed platelet adhesion in the same order as PEG, so similar stealth behaviour can be
assumed.23 Veronese et al. showed compatibility of the poly(oxazoline)s with erythrocytes
and demonstrated that PEtOx 20 kDa is safe and non-toxic when used for intravenous
administration (given every second day during 2 weeks, dosed up to 50 mg/kg). The blood
circulation times were prolonged in the same range as for PEG.24
The cytotoxicity of POx was investigated by examining the cell viability after incubation
with POxs containing different alkyl side chains and with different molecular weights. The
POxs were found to have no influence on the cell growth and proliferation.25 Another point
of investigation was the effect of aromatic oxazoline polymers and PEtOx with
FIGURE 1.3: γ-camera imaging of in vivo distribution of PMeOx48PipDOTA[111In] in a CD1 mouse 30 min and 3 h after intravenous injection. Areas with highest activity concentration are the bladder (thin arrowhead), the kidneys (arrows) and the blood pool in the heart (thick arrowhead).
5
monomer/initiator ratio of 100 on the immunological activity of macrophages. Both were
found to be safe and biocompatible, because no immunosuppressive or undesirable effects
on the macrophage activity were seen. In addition, Luxenhofer et al. investigated relative
toxicity of POx homo- and block copolymers also by means of cell viability. They
demonstrate that the POxs are generally well tolerated by mammalian cells.26
Nevertheless, biocompatibility is not the only important aspect. Once coupled to an
API (e.g. an enzyme) the polymer may not interfere with the effect of the drug. Saegusa et
al. were the first to report about this issue.27 They demonstrated that the enzyme activity of
bovine liver catalase coupled to PMeOx was influenced by the molecular weight of the
polymer and the extent of modification. Once attached to the polymer, the API must keep its
activity as high as possible. Enzymes and insulin conjugated with POx were also investigated
by Viegas and co-workers.28 The conjugated enzymes were tested for in vitro activity and the
insulin conjugates were tested in rats for glucose lowering activity, where they showed
comparable activity as enzymes conjugated with PEG. Again, it was found that the enzyme
activity depends on the extent of protein modification. The activity of insulin remained after
conjugation.
Enzyme-POx-conjugates were also compared to enzyme-PEG-conjugates by Hoogenboom,
Veronese et al. for enzymatic activity.29 Coupling of PEtOx to trypsine had no influence on
the enzyme activity on low molar mass substrates, but reduced the activity on higher molar
mass substrates.
Besides for the purpose of conjugating to a drug or a biomolecule, poly(2-oxazoline)s
can be used for functionalizing liposomes or micelles to deliver drugs. POx-functionalized
liposomes are comparable to PEGylated liposomes with regard to the beneficial properties
that can be attained, e.g. long plasma lifetimes and low hepatosplenic uptake (uptake by
liver and spleen).20,21
The use of micelles made of PEtOx-block-poly(ε-caprolactone) to deliver paclitaxel, a poor
watersoluble drug, was investigated by Jeong and co-workers.30 The micelles had similar in
vitro inhibition of carcinoma cells while exhibiting low cytotoxicity and furthermore
suppressing hypersensitivity and neurotoxicity.
These investigations were extended by Luxenhofer et al., who studied amphiphilic di- and
triblock copolymers based on POx for the delivery of paclitaxel, amphotericin B and
6
cyclosporine A, three poor water soluble drugs.31 The formed micelles showed a high
capacity for drug solubilization. Together with the easy synthesis, low toxicity and other
good properties, it makes poly(2-oxazoline)s ideal for use in drug delivery.
Furthermore, Wang and Hsiue used a triblock copolymer that forms micelles to deliver
doxorubicine.32 The copolymer is temperature- and pH-sensitive due to a middle block of
PEtOx. This pH-responsivity can be used for targeted drug delivery in cancer cells, because
they are more acidic than normal cells.
To summerize, the easy synthesis, biocompatibility and stimuli responsiveness of POxs
make them ideal candidates to be used as carriers in drug delivery applications.
Why is it so interesting to investigate poly(2-oxazoline)s as an alternative to PEG? As
mentioned before, there are numerous drawbacks to PEG.4 In addition, POxs have some very
favourable properties. Or, to cite R. Luxenhofer et al.31 “We believe that the facile synthesis,
excellent water solubility and high loading capacity in combination with formulation stability,
low toxicity, limited complement activation and excellent preliminary in vivo drug efficacy
makes such poly(2-oxazoline)s excellent candidates for further investigations, especially, but
not only in the context of drug delivery.”
Table 1.1 contains an overview of the differences in properties between PEG and POx.
TABLE 1.1: Differences in properties between PEG and POx (adopted from reference 28).
Difficult polymerization process Easy synthesis, with standard glassware and nonexplosive materials
Forms peroxides, antioxidant required Doesn’t form peroxides
Only stable at -20°C Stable at room temperature and in water
Diol content of 2-6% No diol
High viscous when in aqueous solutions Low viscosity
Low drug loading High drug loading due to side chains
Difficult to actively target Active targeting possible with pendent polymers
Can accumulate in some organs and form vacuoles because of desiccant nature of PEG. It forms crystal structures.
Readily cleared from the body and is not hygroscopic. Doesn’t form crystal structures.
7
An interesting structural property of poly(2-oxazoline)s is that they bear an amide
group as side chain on each third atom of the polymer backbone (see Fig.1.4, right). This side
chain can be altered by varying the group at the 2-position in the corresponding monomer
(see Fig.1.4, left). In this way, different properties can be obtained by changing the side
chain.33,34,35 One of these properties is the water solubility. For example, PMeOx and PEtOx
are hydrophilic polymers, but poly(2-butyl-2-oxazoline) or poly(2-phenyl-2-oxazoline) are
hydrophobic. A combination of a water soluble and a water insoluble monomer in a block
copolymer results in an amphiphilic co-polymer. By synthesizing block copolymers of
monomers with different water solubility, micelles and vesicles can be formed.
FIGURE 1.4: Left: 2-oxazoline monomer; Right: General structure of the poly(2-oxazoline)s
Another property that can be finely tuned in this way is the thermoreponsitivity. In
addition, POxs are regarded as “smart polymers” that undergo a change in solution
properties upon a change of an external stimulus. The most investigated responsive
behaviour of POxs is thermosensitivity.36,37,38 Aqueous solutions of PEtOx were firstly
reported in 198839 to exhibit Lower Critical Solution Temperature (LCST) behaviour. This
means that the polymer is water soluble below a certain temperature (the cloud point) and
collapses undergoing precipitation above it. This behaviour is common with other POxs like
Poly(2-n-Propyl-2-oxazoline)s and the transition temperature can be finely tuned by
variation of the polymer length and by copolymerization.40
This interesting smart behaviour of POxs has been exploited for the preparation of
thermoresponsive hydrogels17, in separation sciences41,42 and especially for the development
of drug delivery applications.43,44,45,46
8
Different POxs exhibit different transition temperatures. In the case of poly(nPrOx) and
poly(iPrOx) the transition temperature is situated in the range of the body temperature, and
therefore these polymers have high potential for the use in biomedical applications.
Another property of amphiphilic (co-)poly(2-oxazoline)s is their ability to self-
assemble.17 This means that the polymer forms an ordered structure out of an unorganized
system, due to specific, local interactions. This can be used for numerous applications. An
overview of these applications is beyond the scope of this introduction, and the reader is
directed to the review developed by R. Hoogenboom.17
Moreover, the end-group functionality of POxs can be tailored by the choice of
initiator and termination agent, allowing for the obtention of telechelic polymers
(prepolymers that can be further polymerized through its reactive end-groups) in a
straightforward manner. Thus, the polymer can bear a drug, a targeting group, an anchoring
group, etc.47 It also makes further post polymerization functionalization possible, including
click chemistry strategies17,28 and poly(2-oxazoline)s bearing quaternary ammonium groups.
These polymers exhibit antimicrobial properties against S. Aureus, as has been shown by
Waschinski and Tiller.48
As explained above, an 2-oxazoline (and thus the corresponding poly(2-oxazoline)) can
be functionalized by modifying the side chain in the 2-position. However, there is another
position that can be used to add an extra side chain: the 4- and the 5-carbon.33 In this way,
at one polymer more than one drug molecule can be attached, so less polymer is needed
compared to when only one drug can be coupled to the chain ends (which is the case with
PEG).
Another way to change properties of the polymer, besides varying the side chain, is
changing the backbone. This can be achieved by using a monomer with a bigger ring.33 The
group of monomers derived from the oxazolines, containing one carbon extra in the ring
(and hence in the backbone of the corresponding polymer), are the oxazines (general
structure: see Fig.1.5). The monomers are all six-membered rings, with an additional carbon
atom compared to 2-oxazolines.
One of the properties that can be changed is the thermosensitivity. In the POx-group, only
PMeOx is always water soluble under ambient pressure, PEtOx has an LCST of about 65°C. In
9
the set of poly(2-oxazine)s PMeOz, PEtOz and PPrOz, both PEtOz and PPrOz are
thermosensitive, although PEtOz only with M/I >50.38
FIGURE 1.5: Left: 2-oxazine monomer; Right: General structure of the poly(2-oxazine)s
Also, the water solubility can be changed by adding a methylene group in the main
chain. Adding a methylene group in each repeating unit of the backbone of PEtOx generates
PEtOz and decreases the water solubility. PEtOz is a structural isomer of PPrOx. The shorter
main chain should result in a better water solubility and higher cloud point temperature.
Instead, the temperature at which PPrOx becomes thermosensitive is lower than that of
PEtOz, because the longer side chain (a propyl group instead of an ethyl group) ‘overrules’
the effect of the shorter backbone. In this way, Schubert, Hoogenboom et al. demonstrated
that a change in the side chain has a bigger influence on the water solubility than a change in
the main chain.38 This can be explained by the fact that if the polymers are dissolved in
water, they are hydrated on the basis of hydrogen bonds established between the amide
hetero atoms and the water molecules. The side chains are directly exposed to the solvent,
thus having more influence on the water solubility of the polymer. The backbone is shielded
from the surrounding medium by the hydrophilic side chain groups on the amide, having less
influence on the water solubility or LCST-behaviour.
The change in water solubility due to the insertion of a methylene group is interesting
related to stealth behaviour. Compared to PEG, PMeOx is better watersoluble, and PEtOx
has the same water solubility. To obtain good stealth behaviour, the polymer should be well
soluble in water. However, the polymer may not be too hydrofilic, otherwise the coupling to
the drug cannot be carried out in organic solvents. Because the drug is water insoluble, the
coupling reaction has to take place in a less polar solvent. If the polymer is too water soluble,
as in the case of PMeOx, it is not soluble in the solvent. Since the insertion of a methylene in
the main chain of the POx decreases the water solubility of the polymer, PMeOz will be less
10
water soluble than PMeOx. The hypothesis is that the water solubility (and also the
hydrophobicity) of PMeOz lies in between PMeOx and PEtOx. Hydrophobicity of POxs was
investigated by Viegas et al. with RP-HPLC.28 They found that the retention time of PEG fell
between those of PMeOx and PEtOx, for different lengths of polymers as shown in Fig.1.6.
FIGURE 1.6: Correlation of the hydrophilicity-lipophilicty balance of PEG, PMeOx (PMOZ in graph), PEtOx (PEOZ in graph), and PPrOx (PEOZ in graph) as determined by reverse phase chromatography. Reprinted from reference 28.
This technique is interesting to compare the hydrophobicity of PMeOz to that of
PMeOx, PEtOx and PEG, hence investigating the upper mentioned hypothesis. If the
hypothesis is true, the coupling of PMeOz to an API will probably result in better stealth
behaviour than PEG and PEtOx due to the better water solubility, and as a result of its higher
lipophilicity, it will be better soluble than PMeOx in the solvent for the coupling reaction.
11
2. OBJECTIVES
Poly(2-oxazoline)s (POxs) are good alternatives for poly(ethylene glycol) (PEG) to
attach to drugs, since they provide stealth behaviour and hence a prolonged residence time
in the blood stream. For good stealth behaviour, the polymer has to be hydrophilic and
resemble the water structure in the hydrated state. However, it may not be too hydrophilic,
otherwise the polymer is not soluble enough in the organic solvent where the coupling
reaction with the drug takes place. This solvent is usually an organic solvent since the drugs
are mostly hydrophobic and the formation of the polymer-drug conjugate is carried out in
solution.
Good hydrophilic polymer candidates in the POx-group are poly(2-methyl-2-oxazoline)
(PMeOx) and poly(2-ethyl-2-oxazoline) (PEtOx). PMeOx is highly water soluble, but cannot
easily be coupled to a hydrophobic drug in organic solution. PEtOx, with an additional carbon
in the side chain, has a solubility in water comparable to PEG, so it would, in principle, not
provide better stealth behavior. Hence, there is still a search for a polymer with a level of
hydrophilicity in between that of PEtOx and PMeOx providing better stealth behaviour than
PEG while still being sufficiently soluble in organic solvents. Considering that changing the
side chain gives a too big difference, changing the main chain could be a better strategy,
since it was recently shown that a change in the main chain influences the properties of the
polymer less than changing the side chain. In this way, the hydrophobicity of PMeOx could
be increased by adding a third methylene group in each repeating unit of the backbone. This
can be accomplished by polymerization of 2-methyl-2-oxazine.
Although there is already some research carried out on poly(2-oxazine)s, this is a
hypothesis yet to be confirmed. This is the ultimate goal of this thesis: to determine and
compare the water solubility properties of these three poly(cyclic imino ether)s, named
PMeOx, PEtOx and PMeOz.
However, first, the three polymers should be synthesized. Therefore, the monomers
are required in pure form. The monomers 2-methyl-2-oxazoline and 2-ethyl-2-oxazoline are
commercially available, but the 2-methyl-2-oxazine has to be synthesized first. Different
synthesis routes will be compared and evaluated.
12
To compare the properties of polymers with different molecular weights, each
polymer will be synthesized with three different chain lengths. The chain length is expressed
by the degree of polymerization (DP), and is determined by the concentration ratio of
monomer and initiator (M/I-ratio). All polymers will be synthesized with a M/I of 50, 100 and
200.
Before preparing these polymers, polymerization kinetic studies have to be carried out
for the three monomers investigated. These studies are required to show the living nature of
the cationic ring opening polymerization, by the obtention of first-order kinetic plots and a
linear increase of molecular weight with conversion. Furthermore, the polymerization rate
constants are required to calculate the required reaction times for preparing the polymers
with different chain lengths.
Once the polymers of different chain lengths are synthesized, the following
characterization should be carried out: Gas Chromatography to determine the conversion,
Size Exclusion Chromatography to determine the polydispersity index (PDI) and the number-
average molecular weight (Mn), and proton Nuclear Magnetic Resonance spectroscopy to
confirm the structure and evaluate the purity.
The water solubility properties, i.e. hydrophilicity, will be investigated by means of
viscosimetry and High Performance Liquid Chromatography for which the methodologies
have to be developed first.
13
3. MATERIALS AND METHODS
3.1. MATERIALS
3.1.1. Monomers
2-Ethyl-2-oxazoline 98% (Aldrich, Steinheim, Germany)
2-Methyl-2-oxazoline 98% (Aldrich, Steinheim, Germany)
3.1.2. Chemicals for the synthesis of the 2-methyl-2-oxazine monomer
3-Amino-1-propanol 99% (Acros, Geel, Belgium)
3-Chloropropylamine hydrochloride (Aldrich, Steinheim, Germany)
Acetic anhydride 99+% (Acros, Geel, Belgium)
Acetonitrile (Sigma Aldrich, Steinheim, Germany)
18-Crown-6-ether (Merck-Schuchardt, Hohenbrunn, Germany)
Silicium oxide (chromatographic silica media 200 micron, Davisil®, Grace Davison,
Worms, Germany)
Tetrahydrofuran 99,5% extra dry over molecular sieves (Acros, Geel, Belgium)
Zinc acetate (Merck, Darmstadt, Germany)
3.1.3. Chemicals for the polymer synthesis
Acetonitrile, 99,9% extra dry over molecular sieves (Acros, Geel, Belgium)
Barium oxide (Aldrich, Steinheim, Germany)
Methyl-p-toluenesulfonate (Aldrich, Steinheim, Germany)
Sodium bicarbonate (Sigma Aldrich, Steinheim, Germany)
3.1.4. Chemicals for evaluation of the watersolubility-related properties
Methanol (Sigma Aldrich, Steinheim, Germany)
PEG 6000 (Uniquema, Middlesbrough, UK)
Poly(ethylene glycol) average M.W. 1500 (Janssen Chimica, Geel, Belgium)
Polyethylenglycol 5000 zur synthese (Merck-Schuchardt, Hohenbrunn, Germany)
14
Polyethylenglykol 10000 (Aldrich, Steinheim, Germany)
3.1.5. General solvents and salts
Dichloromethane (Sigma Aldrich, Steinheim, Germany)
Diethyl ether (Sigma Aldrich, Steinheim, Germany)
Ethyl acetate (Sigma Aldrich, Steinheim, Germany)
Hexane (Fisher Scientific, Loughborough, UK)
Magnesium sulfate (Sigma Aldrich, Steinheim, Germany)
Potassium hydroxide (Fisher Scientific, Loughborough, UK)
Sodium sulfate (Sigma Aldrich, Steinheim, Germany)
Sodium carbonate (Sigma Aldrich, Steinheim, Germany)
3.1.6. Devices
Microwave reactor: Anton Paar monowave 300, autosampler: MAS 24
Size Exclusion Chromatography (SEC):
- DMA-SEC 1: Agilent 1260-series equipped with a 1260 ISO-pump, a 1260 Diode Array
Detector (DAD), a 1260 Refractive Index Detector (RID), and a PSS Gram30 column in series
with a PSS Gram1000 column inside a 1260 Thermostated Column Compartment (TCC) at
50°C. N,N-Dimethylacetamide (DMA) containing 50 mM of LiCl was used as eluent, the flow
rate was 1 ml/min. Calibration standards: Poly(methyl methacrylate) (PMMA).
- DMA-SEC2: Waters 600 Controller, pump: Waters 610 Fluid Unit 1 ml/min, Waters 2414 RI
Detector, Merck Hitachi column oven L-7300 at 40°C, Waters 717 Plus Autosampler
Empower Software, Eluent was N,N-dimethylacetamide of Sigma-Aldrich ref. D137510,
columns were: 1 x GPC precolumn PSS GRAM analytical 10µm 8,0x50 mm, 1 x GPC column
PSS GRAM analytical 30 A°, 10 µm 8,0x300 mm, 2 x GPC column PSS GRAM analytical 1000
A°, 10 µm 8,0x300 mm. Calibration standards: Poly(methyl methacrylate) (PMMA).
- HFIP-SEC: system equipped with a Waters 1515 Isocratic HPLC pump, a Waters 2414
refractive index detector (40°C), a Waters 2707 autosampler, and a PSS PFG guard column
followed by 2 PFGlinear-XL (7 mm, 8 x 300 mm) columns in series at 40°C.
15
Hexafluoroisopropanol (HFIP, Apollo Scientific Limited) with potassium trifluoroacetate (3 g
.L-1) was used as eluent at a flow rate of 0,8 mL.min-1. The molar masses were calculated
against polystyrene standards.
Gas Chromatography (GC):
Agilent 7890A equipped with FID detector. Stationary phase was HP-5, mobile phase H2
(flow: 1,5 ml/min). Column: length=30 m, diameter=0,32 mm, film=0,25 µm. Thermal
gradient: start at 50°C, heating to 120°C with a rate of 20°C/min (hold time 3,5 min), further
heating to 300°C with a rate of 50°C/min (hold time 7,6 min).
Viscosimeter:
Vibro viscosimeter SV-10 from AND A&D Company Limited. Range: 0,3-10000 mPa.s. Line-
way Vibro viscosimeter using tuning-fork vibration method (vibration frequency: 30 Hz).
Repeatability: 1% (standard deviation). Glass sample cup AX-SV-35 was used.
High Performance Liquid Chromatography (HPLC):
Agilent 1100 with autosampler, quaternary pump, methanol:water 90:10 used as eluent
(brand, isocratic), UV detection (DAD) at 214,16 nm, 35°C, flow: 0,8 ml/min, injection
volume: 25 µl, sample concentration: 1 mg/ml, run time: 15 min. Column: Waters XBridge
C18, 3,5 µm, 100 Å pore size.
NMR:
Bruker AVANCE 300 MHz. Three-channel spectrometer, 5mm BBO probe with ATM, BACS-60
Sample changer for 60 samples, z-Pulsed Field Gradients, 2H gradient shimming, Operator
Access and Open Acces, Running Topspin 2.1 and ICONNMR, Involved in last year elective
course ‘Advanced NMR'. Deuterated chloroform (CDCl3) was used as solvent.
Centrifuge ALC multispeed (thermo stated), PK 121R
3.2. METHODS
3.2.1. Distillation
All glass parts of the distillation setup were dried in the oven at 140°C and heated up
with the heat gun under vacuum (oil pump) after building up the installation.
16
MeOx and EtOx were dried over barium oxide for half an hour before distillation. The
distillation was carried out under dry Argon. For MeOx, the distillation was carried out at
147°C, for EtOx at 165°C (both external oil bath temperature).
The initiator, methyl-p-toluenesulfonate (methyl tosylate, MeOTs) was distilled under
reduced pressure. No drying agent was used prior to the distillation, because the product of
the reaction between barium oxide and water, barium hydroxide, reacts with MeOTs.
Molecular sieves were used to prevent boiling delays of the MeOTs during distillation. The
temperature at which the MeOTs was collected was 141°C (oil bath) and 95°C
(thermometer), discarding the first and the last fraction of the distillation.
3.2.2. Synthesis of the 2-methyl-2-oxazine
Two routes were explored for the synthesis of the 2-methyl-2-oxazine.
First route: ACN (3 eq.; 504,4 mmol; 20,9 g) and zinc acetate (0,05 eq.; 8,4 mmol; 1,5 g)
were heated up to 90°C and 3-amino-1-propanol (1 eq.; 168,1 mmol; 13,5 g) was added
drop wise. After 24 h refluxing, the reaction mixture was allowed to cool down to room
temperature and 100 ml dichloromethane (DCM) was added. The resulting organic phase
was washed four times with 50 ml of water, and one time with 50 ml of brine. Then the
organic phase was dried over magnesium sulphate and the DCM evaporated in the
rotavapor. Yield: 16,8%, 3,0 g.
1H NMR (300 MHz, δ in ppm, CDCl3): 4.10 (t, CH2O, 2H), 3.3 (t, CH2N, 2H), 1.90 (m,
CH2CH2CH2; CH3, 5H).
The second route is a two-step reaction.
First step: to a cooled solution (5°C) of 3-chloropropylamine hydrochloride (43,7 g; 336,2
mmol) and acetic anhydride 99+% (34,3 g; 336,2 mmol) in distilled water (336ml), sodium
bicarbonate (70,6 g; 840,6 mmol) was slowly added. The solution was allowed to stir for 10
minutes at 5°C. Three extractions were carried out with ethyl acetate: the first with 0,5 l, the
second with 0,2 l and the third with 0,3 l. The organic ethyl acetate-phase was dried over
sodium carbonate and the solvent was evaporated. The product (N-(3-
chloropropyl)acetamide) was purified with silica column chromatography with
17
cyclohexane/ethyl acetate (1:1) as eluent. The column chromatography was followed by
taking fractions and measure them by thin layer chromatography (TLC) (staining agent:
potassium permanganate). Yield: 11,5%, 5,2 g
1H NMR (300 MHz, δ in ppm, CDCl3): 5.90 (bs, 1H), 3.60 (t, 2H), 3.4 (q, 2H), 2.00 (m, 5H).
The ring closure was carried out by dissolving the N-(3-chloropropyl)acetamide (2.7 g;
20 mmol) and 18-crown-6-ether (0,26 g; 1 mmol) in dry tetrahydrofuran (THF) (60 ml) under
argon atmosphere. Subsequently, potassium hydroxide (3,4 g; 60 mmol) was added portion
wise at room temperature. After one hour, the reaction was stopped by adding the mixture
to 100 ml of water. Then, the water phase was extracted with diethyl ether (2x50 ml). The
mixed organic phases were washed four times with 50 ml of water and two times with brine.
The washed organic phase was dried over magnesium sulphate and the solvent evaporated.
Yield: 0%.
3.2.3. Polymerization of 2-oxazolines
The preparation of the reaction mixture was carried out under dry argon atmosphere.
The initiator and the monomer were weighed according to the desired M/I-ratio, and the
acetonitrile (extra dry) was added with a syringe obtaining a total volume of 2,5 ml a
monomer concentration of 4 M. All tools needed for the preparation of the reaction mixture
were dried in the oven at 140°C and allowed to cool to room temperature under argon
atmosphere. The polymerization was carried out in a microwave reactor in capped vials. The
microwave reactor heats up the reaction mixture directly, so the reaction can proceed in a
controlled way at temperatures beyond the boiling point of acetonitrile. The reaction
temperature was set at 140°C, because this was found to be the optimum temperature for
this polymerization.49 The reaction times depend on the concentration of living cationic
chains, assumed to be equal to the initiator concentration. The following reaction times
were used (for both PMeOx and PEtOx), according the article of Schubert et al.49: M/I50: 500
s, M/I100: 1000 s, M/I200: 2000 s.
After the reaction the vials were cooled down by passing a nitrogen stream, and the
reaction was quenched with a saturated sodium bicarbonate solution.
18
The acetonitrile was removed from the quenched polymerization mixture under
reduced pressure, and the residue redissolved in a small amount of dichloromethane. This
solution was added drop wise to cold diethyl ether under vigorous stirring, with the
development of a white precipitate of poly(2-oxazoline). The suspension was centrifuged at
0°C for 5 minutes with 5000 rotations per minute. The precipitate was transferred with
dichloromethane in a vial and the solvent was evaporated, obtaining a white powder. The
vial was further dried under vacuum (vacuum pump and vacuum oven).
The polymers were characterized by means of 1H NMR spectroscopy and size exclusion
chromatography (SEC). Conversion was measured by gas chromatography (GC).
3.2.4. Kinetic studies
Kinetic studies were carried out for the polymerization of MeOx, EtOx and MeOz. The
formation of polymers with a M/I of 100 was chosen, because these polymers are situated
between the M/I50- and M/I200-polymers, and hence supposed to be representative for all
polymerizations aiming for different chain lenghts. All reactions were carried out at 140°C.
Firstly, a stock solution was made according to the procedure described in the previous
section. Then, several vials were prepared containing 600 µl of the stock solution. Each vial
was allowed to react for a different time at 140°C in the microwave reactor to reach a
different level of conversion. Al the reactions were quenched by adding a drop of water. The
polymers were characterized by means of 1H NMR spectroscopy and size exclusion
chromatography (SEC). Conversion was measured by gas chromatography (GC).
3.2.4.1. Polymerization kinetics of poly(2-methyl-2-oxazoline)
A 3,5 ml stock solution was prepared, with M/I=100 and [MeOx]=4 M.
The vials were allowed to react for following times: 75 s, 150 s, 300 s, 450 s, 600 s. So, five
data points were obtained. At 600 s, the reaction theoretically reaches 99% conversion.
19
3.2.4.2. Polymerization kinetics of poly(2-ethyl-2-oxazoline)
A 3,5 ml stock solution was prepared, M/I=100 and [EtOx]=4 M. The reaction times
were: 90 s, 210 s, 330 s, 450 s, 580 s, 700 s. At 700 s, the reaction theoretically reaches 99%
conversion.
3.2.4.3. Polymerization kinetics of poly(2-methyl-2-oxazine)
A vial was prepared with methyl tosylate (37.7 mg, 203.5 μmols, 0.01 equiv),
acetonitrile (4.2 mL) and 2-methyl-2-oxazine (2.102 mL, 1 equiv). Reaction times were for 5
min, 10 min, 15 min, 20 min, 30 min and 45 minutes. The kinetic experiments were carried
out by dr. Bryn Monnery.
20
4. RESULTS AND DISCUSSION
To evaluate poly(2-methyl-2-oxazine) as a possible stealth polymer, water solubility
related properties have to be measured and compared to poly(2-methyl-2-oxazoline) and
poly(2-ethyl-2-oxazoline).
Therefore, initiator and monomers were needed in pure form (obtained by
distillation), then the polymers could be synthesized and their reaction kinetics determined.
4.1. PURIFICATION OF MEOTS, MEOX AND ETOX
Before distillation, the two commercial available monomers (2-methyl-2-oxazoline and
2-ethyl-2-oxazoline) were dried over barium oxide. Methyl tosylate was distillated directly.
Distillation of the monomers were carried out under argon atmosphere, which means
that the distillation setup had to be connected to a Schlenk line that was provided with an
argon flow.
Distillation of the initiator (MeOTs) was carried out under reduced pressure. The
distillation setup was connected to a Schlenk line provided with a vacuum pump.
4.2. SYNTHESIS OF THE 2-METHYL-2-OXAZINE
Two routes were explored for the synthesis of the 2-methyl-2-oxazine. The first route
is the cyclo condensation of an amino alcohol and a nitrile described by Ritter et al.50 The
second route is a two-step reaction and happens via an acid amide.
4.2.1. Cyclo condensation of 3-amino-1-propanol and acetonitrile
It follows this reaction scheme:
21
Condensation of acetonitrile with an amino propanol results in ring closure with release of
ammonia.
The mechanism is depicted hereafter:
Zinc acetate pulls the electrons out of the triple bond facilitating the attack of the
amine of 3-amino-1-propanol at the triple bonded carbon in the nitrile. After the attachment
of the amino propanol via the amine, the hydroxyl group will attack the same carbon of
acetonitrile, resulting in ring closure and release of ammonia.
After three attempts, 3 g MeOz was obtained (a yield of 16,8%). Unfortunately, this
was not enough to distil and use for polymerizations. A possible explanation for the low yield
is the low temperature used, which is limited by the boiling point of acetonitrile. A better
yield is normally obtained when such cyclo condensations are performed with temperatures
up to 130°C, as e.g. described by Kim et al.51 Nonetheless, the monomer was quite pure,
according to the 1H NMR spectrum showed in Fig. 4.1.
22
FIGURE 4.1: 1H NMR spectrum of MeOz (CDCl3 as solvent).
Besides the expected signals, only a minor acetonitrile (ACN) residue is observed. The CHCl3
is present in the utilized CDCl3 NMR solvent.
4.2.2. Synthesis of MeOz via the acid amide
This is a two-step reaction. After the first step, an acid amide is formed by reaction of
an amine group with acid anhydride. In the second step, ring closure continues, obtaining
the 2-methyl-2-oxazine.
First step: reaction scheme:
23
Reaction mechanism:
Sodium bicarbonate is added to obtain the uncharged 3-chloropropylamine with a reactive
amine group. Attack of the amine group to the acid anhydride yields 3-
chloropropylacetamide by emitting acetic acid.
Second step: reaction scheme:
Reaction mechanism:
Potassium hydroxide is not soluble in tetrahydrofuran (THF, solvent). Therefore, 18-crown-6-
ether is added to capture the potassium-ion, enhancing the solubility of KOH in THF. KOH
deprotonates the nitrogen, yielding a negative charge at the oxygen. By attack of this
negatively charged oxygen on the 3-carbon and emitting the chloride, ring closure is
obtained.
24
This route only yielded a small amount of monomer. The yield of the first step was
11,5%, but the yield of the second step was almost 0%. After the first step, a pure product
was obtained, according to the 1H NMR spectrum shown in Fig. 4.2. A possible reason for
unsuccessful ring closure is that the potassium hydroxide was used as pellets, and hence
could not react properly because they were not enough dissolved despite the presence of
18-crown-6-ether.
FIGURE 4.2: 1H NMR spectrum of 3-chloropropylacetamide (CDCl3 as solvent)
Due to time constraints for the thesis, it was not possible to further optimize the
monomer synthesis procedure, although it could be concluded that the first route is more
promising. Fortunately, Dr. Bryn Monnery was able to synthesize the monomer via the first
route by using an oil bath temperature of 130°C, towards the end of my thesis, and hence
kinetic experiments could be carried out.
4.3. POLYMERIZATION: GENERAL ASPECTS
Only the poly(2-oxazoline)s were synthesised and purified. The poly(2-methyl-2-
oxazine) from an earlier study was used, there was no time anymore to make it.
25
4.3.1. Reaction mechanism
FIGURE 4.3: Reaction mechanism of the polymerization of oxazolines.
The living cationic ring opening polymerization reaction, as depicted in Fig. 4.3,
comprises three phases: initiation, propagation and termination. As depicted in the same
figure, a nucleophile can be used to introduce a chain end functionality, but the presence of
unwanted nucleophiles also terminates the reaction. Water is a good nucleophile, so it
terminates the reaction by adding a hydroxyl group to the chain. Moreover, the remaining
proton initiates a new chain as it contains a positive charge. The occurrence of such chain
transfer reactions causes a broader molecular weight-distribution, resulting in a higher
polydispersity and loss of chain-end functionality. Because of these reactions, the
polymerization has to be done completely free of water, even moist from the air.
As a side reaction, the nucleophile can attack the living chain at the 2-position, as
depicted in Fig.4.4. This results in an ester as end group and is seen in the NMR spectra.
26
FIGURE 4.4: Side reaction of the POx-polymerization.
4.3.2. Determination of the monomer conversion
A measure for the progress of the polymerization reaction is the monomer conversion.
This was measured by means of gas chromatography (GC). GC is a chromatographic
technique that uses a solid as stationary phase and an inert gas as mobile phase. This
technique uses elevated temperature and is therefore suitable to measure volatile
components in a sample. After injection, the components evaporate depending on their
vapour pressure, are subsequently mixed and taken with the carrier gas through the column
and separated on basis of their affinity for the stationary phase. For each polymer, a sample
for GC was taken before and after the polymerization, and the peaks correspondent to
acetonitrile and monomer were integrated. The retention time of acetonitrile was around
1,4 minutes, the monomers eluted from the column after about 1,9 min. The conversion was
calculated from the difference in monomer/solvent ratio between the sample at a specific
time RA,t and the ratio at time zero, RA,0 as shown in the equation.
with
4.3.3. Characterization
1H NMR spectroscopy gave us information on the composition and purity of the
obtained poly(2-oxazoline).
27
The molecular weight distribution was investigated by SEC. SEC is a chromatographic
method in which molecules are separated by their hydrodynamic volume (hydrated size in
solution). The stationary phase is made of microporous gasket material with a certain pore
size. Small molecules are retained as they pass through all pores, big molecules pass along
the packing material and are not retained. The parameters studied were the number
average molecular weight (Mn), and the polydispersity index (PDI). The PDI is calculated by
dividing the weight-average molecular weight (Mw) by the number-average molecular weight
(Mn). A PDI of 1 is ideal, meaning that all polymers have exactly the same number of
repeating units, a PDI lower than 1,3 is indicative of a living/controlled polymerization and is
sufficient for the application in this thesis work.
4.4. KINETIC STUDIES
Because the polymerization reaction is first order in cationic propagating species, the
rate of the reaction is given by the following formula:
where [M]= monomer concentration (mmol.ml-1)
kp= rate constant (l.mol-1.s-1)
[P+]= active living species (mmol.ml-1)
After integration we obtain:
where [M]0= initial monomer concentration (mmol.ml-1)
[M]t= monomer concentration at time t (mmol.ml-1)
kp= rate constant (l.mol-1.s-1)
[P+]= active living cationic species (mmol.ml-1)
t= time (s)
Because each active living species (growing chain) is initiated by initiator, one can say
that [P+]=[I]0, when assuming fast and complete initiation. Hence we get:
28
where [M]0 = initial monomer concentration (mmol.ml-1)
[M]t = monomer concentration at time t (mmol.ml-1)
kp= rate contstant (l.mol-1.s-1)
[I]0 = initiator concentration (mmol.ml-1)
t= time (s)
As the polymerization indeed follows first-order kinetics,
plotted against time
should give a straight line with kp.[I]0 as slope if there is no termination during the
polymerization. This is plotted in each first graph (Fig.4.5 and Fig.4.7).
represents
the monomer conversion and was measured by GC. In this way, the monomer conversion is
plotted against time. Values higher than 4 were not depicted in the graph, because the
conversion is already higher than 99% resulting in inaccuracies due to reaching the limits of
GC sensitivity.
The livingness of the polymerization reaction can be proven by the linear first-order
kinetics together with a linear increase of the number-average molecular weight with
conversion (not for high conversions). In an ideal living polymerization all chains start
growing at the same time and they all keep on growing with the same rate. Hence, their
length is determined by conversion. If chain transfer occurs, a negative deviation from
linearity would be observed. This is depicted in Fig.4.6 and Fig.4.8. The dotted line shows the
variation of the theoretical molecular weight in function of conversion. The theoretical
molecular weight was calculated by this formula:
Conversion x DP x MWmonomer + MWinitiator
where DP = degree of polymerization
MWmonomer = molecular weight of the monomer polymerized (g/mol)
MWinitiator = molecular weight of the initiator used (g/mol)
29
4.4.1. Kinetic studies of PMeOx
FIGURE 4.5: 0
plotted against time for the polymerization of MeOx with DP = 100.
The graphs does not pass the point 0,0. This is can be due to chain transfer, giving a
negative deviation.
As mentioned above, the rate constant kp can be calculated from the slope. As a
monomer concentration of 4 M and a M/I ratio of 100 is used, the initiator concentration is
0,04 M. In this way, the kp of the polymerization of MeOx at 140°C with M/I=100, calculated
out of this plot, is 0,085 l.mol-1.s-1. In literature, a rate constant of 0,22 l.mol-1.s-1 is
mentioned.
y = 0,0034x + 0,7276 R² = 0,9494
0
0,5
1
1,5
2
2,5
3
0 100 200 300 400 500 600 700
ln([
M0]/
[Mt]
)
Time (sec.)
Polymerization PMeOx
30
FIGURE 4.6: Molecular weight in function of conversion and PDI in function of conversion for the polymerization of 2-methyl-2-oxazoline. The red dots were not taken into account for the linear fit of the data points, because the reaction did not proceed any more (as there is no increase in molecular weight). The dotted line shows the evolution of the theoretical molecular weight in function of conversion.
It is difficult to draw definite conclusions out of this plot, because no samples were
assayed at low conversion. At high conversions, chain coupling occurs, resulting in a too high
Mn.
31
4.4.2. Kinetic studies of PEtOx
FIGURE 4.7: 0
plotted against time for the polymerization of EtOx with DP = 100.
Calculated from the plot, the rate constant kp is 0,18 l.mol-1.s-1. In literature, a constant
of 0,082 l.mol-1.s-1 is mentioned.
y = 0,0072x - 0,1734 R² = 0,962
0
0,5
1
1,5
2
2,5
3
3,5
0 50 100 150 200 250 300 350 400 450 500
ln([
M0]/
[Mt]
)
Time (sec.)
Polymerization EtOx
32
FIGURE 4.8: Molecular weight in function of conversion and PDI in function of conversion for the polymerization of 2-ethyl-2-oxazoline. The red dots were not taken into account for the linear fit of the data points, because the reaction did not proceed any more (as there is no increase in molecular weight and 99% conversion was reached). The dotted line shows the evolution of the theoretical molecular weight in function of conversion.
The Mn values seem to increase in a controlled way. They are slightly high, possible due
to DMA calibration.
4.4.3. Kinetic studies of PMeOx
These were carried out, but did not gave the desired results. Almost no reaction
occured, because there was still (after several purification attempts) ammonia present. As
this is a nucleophile, it terminates the reaction immediately.
33
4.5. POLYMER SYNTHESIS
4.5.1. Precipitation and characterization
After synthesis, the polymers were purified by precipitation in cold diethyl ether to
remove the initiator, unreacted monomer, solvent and shorter polymer chains. In this way,
polymers with a better poly dispersity are obtained.
After the work up, a sample was measured in the SEC to determine the PDI and the
Mn. Table 4.1 gives an overview of the PDI- and Mn-values of the synthesized polymers.
TABLE 4.1: Overview of the PDI and Mn values of the synthesized polymers after work up, measured in different SEC devices.
Polymer PDI Mn
DMA-SEC 1 HFIP-SEC DMA-SEC 2 DMA-SEC 1 HFIP-SEC DMA-SEC 2
PMeOx50 1,2 1,1 - 6500 6700 -
PMeOx100 1,2 1,2 - 12500 10000 -
PMeOx200 1,3 1,2 - 14000 13000 -
PEtOx50 1,1 - 1,2 8000 - 5000
PEtOx100 1,2 - 1,2 14000 - 10000
PEtOx200 1,2 - 1,1 21000 - 20000
As can be seen, well defined poly(2-oxazoline)s with PDI values below 1,3 were obtained.
The conversion was for all samples more than 97%, the isolated yield was more than 94%.
4.5.2. 1H NMR spectra
From all polymers, a 1H NMR spectrum was recorded. Figures 4.9 until 4.14 show the
1H NMR spectra of all polymers.
Some of the 1H NMR spectra contain a DCM-peak, probably due to contamination in
the NMR solvent. Also some initiator as tosylate anion still present. The peaks indicated by d
and e are due to the side reaction, depicted in Fig.4.4.
34
FIGURE 4.9: 1H NMR spectrum of PMeOx50 (CDCl3 as solvent).
FIGURE 4.10: 1H NMR spectrum of PMeOx100 (CDCl3 as solvent).
35
FIGURE 4.11: 1H NMR spectrum of PMeOx200 (CDCl3 as solvent).
FIGURE 4.12: 1H NMR spectrum of PEtOx50 (CDCl3 as solvent).
36
FIGURE 4.13: 1H NMR spectrum of PEtOx100 (CDCl3 as solvent).
FIGURE 4.14: 1H NMR spectrum of PEtOx200 (CDCl3 as solvent).
37
The degree of polymerization can be calculated from the NMR spectra. Because the
number of initiator-protons in the polymer chain was calibrated as 3, the number of total
backbone protons (a) has to be divided by the number of protons in one repeating unit. For
example, the number of backbone protons for the PMeOx50 is 153 (Fig. 4.9). As there are 4
protons in each repeating unit, a value of 38,25 is calculated for the degree of
polymerization. This value is lower than the expected degree of polymerization of 50
(because of the [M]/[I] ratio that was used by preparation of the polymers and the Mn
value). The reason for the low value of total backbone protons is that the peak shape of
NMR signals is Lorentzian meaning that they are very broad near the baseline. Hence, if the
initiation peak is integrated, a higher value is obtained for the number of protons due to
overlap with the backbone signal resulting in a lower number of backbone protons
compared to the initiator protons in the chain. Therefore, we have not taken these data
further into account.
4.6. WATER SOLUBILITY RELATED TESTS ON THE POLYMERS
4.6.1. Viscosimetry
Measuring a 2% solution of PEG1500 and a 2% solution of PEtOx2000 obtained
inconsistent results. Calibration was carried out with deionised water. After measuring a
sample, the water did not gave back the value at which it was calibrated. On top, measuring
one sample several times gave different values. Therefore, the measurements were not
reliable. A possible explanation is that the polymer concentration was too low.
4.6.2. RP-HPLC measurements of the tested polymers
Measuring polymers by HPLC is not easy, because they get easily stucked in the
column. Therefore, pore size, column material, carbon load and solvent are important to
consider by choosing the column. We choose a column with a quite small pore size, but a
high carbon load to measure our polymers.
As eluent, we used a mixture of water and methanol. To obtain a acceptable retention
time, we chained the solvent, yielding 90:10 methanol:water as most optimal eluent
38
composition. Mixtures containing less methanol gave peak broadening and even several
peaks.
The chromatograms of the different polymers measured are showed hereafter. As can
be seen, the polymers elute very fast over the column. A lot of solvent peaks are located
before 2,5 minutes, and several polymer peaks are very close to this area.
Most of the polymer peaks are broad, this due to high polymer concentrations. Broad
peaks are not desired, because determination of the retention time is more difficult.
PMeOz-samples were obtained from kinetic studies, described in reference 38.
PMeOx50
PMeOx100
m in0 2 4 6 8 10 12 14 16
m AU
0
200
400
600
800
D AD 1 B, S ig=214,16 R ef=off (D :\D ATA\12-05-10\FB000014.D )
1.5
07
1.7
71
2.1
90
2.2
31
3.6
83
m in0 2 4 6 8 10 12 14 16
m AU
0
200
400
600
800
D AD 1 B, S ig=214,16 R ef=off (D :\D ATA\12-05-10\FB000016.D )
1.5
55
1.9
85
2.0
76
2.5
51
3.2
97
39
PMeOx200
PEtOx50
PEtOx100
m in0 2 4 6 8 10 12 14 16
m AU
0
200
400
600
800
1000
D AD 1 B, S ig=214,16 R ef=off (D :\D ATA\12-05-10\FB000017.D )
1.4
84
1.9
75
2.9
30
m in0 2 4 6 8 10 12 14 16
m AU
0
200
400
600
800
1000
1200
D AD 1 B, S ig=214,16 R ef=off (D :\D ATA\12-05-10\FB000015.D )
1.6
89
2.0
23
2.2
80
2.3
32
2.3
83
2.4
33
4.1
22
5.7
81
m in0 2 4 6 8 10 12 14 16
m AU
0
200
400
600
800
1000
D AD 1 B, S ig=214,16 R ef=off (D :\D ATA\12-05-10\FB000019.D )
1.9
02
2.0
45
2.1
06
2.3
79
2.6
59
4.9
22
6.2
45
7.1
49
40
PEtOx200
PMeOz50
PMeOz100
m in0 2 4 6 8 10 12 14 16
m AU
0
200
400
600
800
1000
1200
D AD 1 B, S ig=214,16 R ef=off (D :\D ATA\12-05-10\FB000013.D )
1.0
18
1.8
35
2.2
83
2.3
86
2.4
87
2.6
40
3.1
11
3.8
42
m in0 2 4 6 8 10 12 14 16
m AU
0
200
400
600
800
1000
D AD 1 B, S ig=214,16 R ef=off (D :\D ATA\12-05-11\FB000102.D )
1.6
74
2.2
24
2.3
53
2.5
55
2.6
06
3.0
04
3.6
65
7.4
04
m in0 2 4 6 8 10 12 14 16
m AU
0
200
400
600
800
1000
1200
D AD 1 B, S ig=214,16 R ef=off (D :\D ATA\12-05-11\FB000103.D )
1.8
78
2.2
32
2.5
89
3.0
01
3.8
51
8.1
57
41
PEG1500
PEG3000
PEG6000
PEG10000
The chromatograms of PEG only show solvent peaks. This is caused by the fact that
PEG is not detectable in methanol with UV detector, as both absorb UV light of the same
wave length.
The retention times are summerized in Fig.4.15 for the detectable polymers. As can be
seen, the retention time of PMeOx decreases slightly with increasing degree of
polymerization. As PMeOx is known as a hydrophilic polymer, it will be readily released from
the column, because not much interactions will occur. Longer the polymer decreases the
retention time.
For PEtOx, no clear trend can be seen. For PEtOx50 and PEtOx100, retention time of
increases with increasing molecular weight, but the retention time of PEtOx200 is even lower
than the one of PEtOx50.
The retention time of PMeOz increases slightly by increasing degree of polymerization
from 50 to 100. Furthermore, Fig.4.15 shows that the retention time of PMeOz50 lies just
below the retention time of PMeOx50 and far below that of PEtOx50. For the polymers with
degree of polymerization 100, the retention time lies between the one of PMeOx and PEtOx.
m in0 2 4 6 8 10 12 14 16
m AU
-40
-30
-20
-10
0
10
20
D AD 1 B, S ig=214,16 R ef=off (D :\D ATA\12-05-11\FB000105.D )
1.5
52
2.2
95
2.3
44
2.4
25
2.4
66
2.7
33 2
.77
6
2.9
94
m in0 2 4 6 8 10 12 14 16
m AU
-40
-30
-20
-10
0
10
20
D AD 1 B, S ig=214,16 R ef=off (D :\D ATA\12-05-11\FB000106.D )
1.5
13
1.9
22
2.1
93
2.2
98
2.5
76
2.6
81
2.7
54
2.9
87
m in0 2 4 6 8 10 12 14 16
m AU
-50
-40
-30
-20
-10
0
10
20
D AD 1 B, S ig=214,16 R ef=off (D :\D ATA\12-05-11\FB000107.D )
1.5
08
2.1
10
2.4
32
2.5
12
2.5
45
2.6
23
2.6
58
2.7
86
2.9
91
m in0 2 4 6 8 10 12 14 16
m AU
-40
-30
-20
-10
0
10
20
30
D AD 1 B, S ig=214,16 R ef=off (D :\D ATA\12-05-11\FB000108.D )
1.4
85
1.7
28
2.1
33
2.2
67
2.3
39
2.6
37
2.6
92
2.9
90
42
FIGURE 4.15: Retention time in function of degree op polymerization for PMeOx, PEtOx and PMeOz.
The obtained data suggest that the hypothesis that the water solubility of PMeOz lies
between the one of PMeOx and PEtOx, is right. The retention time of PMeOz lies nearer to
that of PMeOx, which agrees with the findings of Schubert et al. that a change in main chain
only has a small influence on the polymer properties.38
As already mentioned, a point of attention is that the polymers elute very fast over the
column and several polymer peaks are very close to the solvent peak area. Changing the
solvent ratio only gave peak broadening, but no change in the retention times. Therefore,
another solvent or another column could have more chance to enhance the retention times
of the polymers.
It is recommended to use RI detection, so, PEG can be detected and its retention times
compared with POxs and PMeOz. It would make the conclusions more powerful.
Further studies should include also a sample of PMeOz200. In this way, a comparison of
the polymers with DP of 200 can be made and possibly a trend can be seen in the evolution
of retention times with increasing degree of polymerization.
0
1
2
3
4
5
6
7
0 50 100 150 200 250
Re
ten
tio
n t
ime
(m
in)
Degree of polymerization
Retention time in function of degree of polymerization
PMeOx
PEtOx
PMeOz
43
5. CONCLUSIONS
To evaluate the use of poly(2-methyl-2-oxazine) as a stealth polymer some properties
related to water solubility were assayed and compared to poly(oxazoline)s.
To achieve this we synthesised PMeOx, PEtOx and MeOz. Pure oxazoline monomers
and methyl tosylate were obtained by distillation. The 2-methyl-2-oxazine monomer was
synthesised and purified by multiple distillations.
Six reference polymers were synthesized: PMeOx and PEtOx with three different chain
lengths (DP of 50, 100 and 200). The molecular weight distributions (PDI values) of the
polymers were narrow (< 1,3), and numerous average molecular weight values were in
agreement with theoretical value. NMR spectra confirmed the structure and revealed that
the polymers were >99% pure.
Kinetic studies of the polymerization of PMeOx, PEtOx and PMeOz with degree of
polymerization 100 were carried out. For PMeOx and PEtOx, they showed the livingness of
the reaction and the first-order consumption of monomer. For PMeOz, no useful data were
obtained, because too much termination occured due to impurities.
The properties related to water solubility we intended to measure were the viscosity
and hydrophobicity. Viscosity measurements gave inconsistent results. Hydrophobicity was
measured by RP-HPLC. The results suggested that the hydrophobicity (and hence the
hydrophilicity) of PMeOz lies between the hydrophobicity of PMeOx and PEtOx, as
hypothetical, but is nearer to that of PMeOx showing greater influence of the side chain
rather than main chain in solubility. More research is required, but the results are consistent
with our hypothesis.
44
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EVENING LECTURES: INTERNATIONALIZATION @ HOME
1. NICK BARBER – IMPROVING ADHERENCE: FROM RESEARCH TO POLICY TO PRACTICE
In the Victorian age, pharmacists touch patients, they treated patients physically.
Pharmacists were engaged with the people.
Nowadays, people get harm from medicines. This harm is caused by non-adherence
from the patient, errors from health professionals and the molecule itself due to
pharmacoepidemiologic causes.
Pharmacists can play an important role to reduce this harm. Therefore, it should get into
government policy.
I think it is very important that pharmacists take more care about how patients take
their medicine. A lot of harm can be taken away by pharmacists, if they are pointed to it and
if they got the tools for it. It would take away a lot of (easy) work and hence pressure from
the doctors, and give pharmacists back what they’ve lost: care about people.
2. WENDY GREENALL – COUNTERFEIT MEDICINES: PFIZER’S FORENSIC LABORATORIES
In times where internet is an important part of our life, counterfeit becomes very
dangerous, because drugs can be sold with easy access. The difference between counterfeit
and the real drug is big, if you consider that the real drug saves lifes and the counterfeit
medicine kills people. But this difference is not always clear from the outside, and even for
trained people it is most of the time impossible to pick out the counterfeit medicine.
Problems of counterfeit are production in unsanitary conditions, the fact that they look like a
general product and containing no active pharmaceutical ingredient (API), another (toxic)
API or the API in wrong concentration.
I think it’s very important that the governments work together to fight against
counterfeit medicines and help pharmacists and patients to differentiate between a real
drug and the counterfeit medicine. This will keep the drugs that we give to patients safe.
3. ALEXANDER ALEX – COMPUTATIONAL CHEMISTRY IN DRUG DISCOVERY: CAN WE
IMPROVE PRODUCTIVITY AND REDUCE ATTRITION?
Since 1980, the number of new chemical entities per year stays constant, despite
increasing research budgets. Most of the molecules fail in the early stages of development.
This high attrition rate is a big problem in drug research.
15 years ago, attrition rate was mainly caused by problems with the kinetic properties
of the drug. To get the drug into the cell was big issue. about 2004, most of the attrition was
caused by side effects and toxicity. Nowadays, a lack of efficiency is the biggest cause of
attrition.
To make drug research more productive, computational chemistry is an essential tool.
With computational design, computational chemistry and virtual screening methods, a
lot of ‘bad molecules’ with a high risk of attrition can already filtered out. In this way, cycling
time in drug discovery can be reduced and hit finding can be enhanced.
I think that computational chemistry in drug discovery is indispensable. But computers
don’t know everything, and I fear that if research only relies on computational models, a lot
of information will be lost.
4. RICHARD O’KENNEDY – APPLICATIONS OF ANTIBODIES IN THE ANALYSIS OF DRUGS,
DISEASE MARKERS, BACTERIA AND TOXINS
There are three important types of antibodies: polyclonal, monoclonal and
recombinant. The last type is not easy to produce and has a high cost, but has a high capacity
of improvement. This means that it is easy to design it with the characteristic you want.
An important application of antibodies is the use as biomarker by cardiovascular
damage: cardiac troponin I is released from the heart, a specific antibody recognizes it.
Another application could be the detection of listeriosis (caused by the bacteria Listeria
monocytogenes). As target, L. Monocytogenes virulence-associated proteins can be used.
There can be antibodies synthesized that are very sensitive to warfarin, morphine and
aflatoxines. In this way, warfarin can be detected in urine. Morphine, which is the main
metabolite of heroin, can be found in saliva in case of heroin abuse. Aflatoxines are very
toxic secondary metabolites of Aspergillus and are found as contaminants in many foods. An
assay with antibodies is very sensitive to detect these contaminants.
It is amazing how many applications there are for antibodies. It is really worthy to
invest more money in research about antibodies, I think.