Evaluation of poly(2-oxazoline) derivatives as poly...

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.



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.

http://pubs.acs.org/action/showImage?doi=10.1021/bc200049d&iName=master.img-006.jpg&type=master

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).


35


FIGURE 4.12: 1H NMR spectrum of PEtOx50 (CDCl3 as solvent).

36



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


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


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


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


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


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


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


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


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


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


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


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.

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