september 2021 a novel heterobifunctional meso-methyl

Author

Pauline Stadler, BSc

Submission

Institute of Polymer

Chemistry

Thesis Supervisor

Assoz. Univ.-Prof. Dr. Ian

Teasdale

Assistant Thesis

Supervisor

Mgr. Paul Strasser, MSc

September 2021

Green-light photo-responsive drug delivery systems based on a novel heterobifunctional meso-methyl BODIPY derivative

Master’s Thesis

to confer the academic degree of

Diplom-Ingenieurin

in the Master’s Program

Chemistry and Chemical Technology

II Pauline Stadler

SWORN DECLARATION

I hereby declare under oath that the submitted Master’s Thesis has been written solely by me

without any third-party assistance, information other than provided sources or aids have not been

used and those used have been fully documented. Sources for literal, paraphrased and cited

quotes have been accurately credited.

The submitted document here present is identical to the electronically submitted text document.

Place, Date ________________________________________________________________

Signature __________________________________________________________________

III Pauline Stadler

Acknowledgement

In this chapter I want to express my gratitude to all, who have helped and supported me during

this thesis. First, I want to thank the whole institute of polymer chemistry for making the work of

this thesis such a good time. I am so grateful for the opportunity to work in this motivated, friendly

and supportive team. My sincere thanks go to Assoz. Univ.-Prof. Dr. Ian Teasdale for his support

and encouragement throughout my thesis and the possibility to work on this fascinating project.

Special thanks go to Paul Strasser for helping me through obstacles and always keeping me

motivated. Although numerous difficulties appeared during my work, I could always count on your

support, patience and competent guidance, which made it such a pleasure to work on this thesis.

I would also like to express my gratitude to Marina Russo and the whole Department of Chemistry

at the Masaryk University in Brno, Czech republic for making photorelease studies for my work.

Next, I thank Prof. Günther Redhammer, University Salzburg for performing the single crystal X-

ray diffraction measurements.

Furthermore, I want to thank my colleagues and friends, who made studying and lectures much

easier with lots of fun and team spirit. I always enjoy spending time with all of you, making fun and

motivating each other.

Last but not least, I want to thank my family for always supporting me in my decisions, your love

and unconditional support.

http://ustavchemie.sci.muni.cz/?q=en

IV Pauline Stadler

Abstract

Stimuli-responsive polymers are attracting widespread interest nowadays, generally attributed to

their manifold areas of application. In the last few years, they have gained a central role in the

research of drug delivery systems, where especially light as the stimulus has been considered

due to its numerous advantages over other stimuli. Photocleavable protecting groups, known as

photocages, allow for remarkable features including non-invasiveness or precise spatial and

temporal control over the release of bioactive substances. Although this approach is promising, it

suffers from the major drawback that most photocages are reactive towards cell-damaging,

ultraviolet light that restricts its application in biological systems. Recent developments have

considered boron-dipyrromethenes (BODIPYs) as photolabile groups for drug delivery due to their

outstanding properties, reaching from high molar absorption coefficients and quantum yields of

release over chemical and photochemical stability to low toxicity. In the context of this master’s

thesis, the synthesis of a novel heterobifunctional meso-methyl BODIPY and its attachment on a

polymer, as well as the synthesis of a small molecular model compound for photophysical

characterization is presented. The BODIPY compounds were analyzed by nuclear magnetic

resonance spectroscopy (NMR), mass spectrometry (MS), infrared spectroscopy (FT-IR), UV-vis

measurements and X-Ray. The BODIPY containing polymers were further analyzed by gel

permeation chromatography (GPC) and quantified with a calibration on the UV-vis spectrometer.

The release of the caged compound when irradiated with light was as well demonstrated with

UV-vis measurements.

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V

Zusammenfassung

Stimuli-responsive Polymers generieren heutzutage großes Interesse, was im Allgemeinen auf

ihre vielfältigen Anwendungsgebiete zurückgeführt werden kann. In den letzten Jahren haben sie

eine zentrale Rolle in der Erforschung von Systemen zur Medikamentenverabreichung erlangt,

wobei besonders Licht als Stimulus aufgrund seiner zahlreichen Vorteile gegenüber anderen

Stimuli berücksichtigt wurden. Photoprotecting groups, bekannt als Photocages, haben

bemerkenswerte Eigenschaften, wie Nicht-Invasivität sowie präzise räumliche und zeitliche

Kontrolle über die Freisetzung gekoppelter bioaktiver Substanzen. Obwohl dieser Ansatz

vielversprechend wäre, ist dessen Anwendung in biologischen Systemen einschränkt, da die

meisten Photocages gegenüber zellschädigendem ultraviolettem Licht reaktiv sind. Neuere

Entwicklungen beschäftigen sich mit Boron-Dipyrromethenes (BODIPYs) aufgrund ihrer

herausragenden Eigenschaften als photolabile Gruppen für den Wirkstofftransport. Diese reichen

von hohen molaren Absorptionskoeffizienten und Quantenausbeuten der Freisetzung über

chemische und photochemische Stabilität bis hin zu geringer Toxizität. Im Rahmen dieser

Masterarbeit wird die Synthese eines neuen hetero-bifunktionellen meso-Methyl-BODIPYs und

dessen Bindung an ein Polymer sowie die Synthese einer kleinmolekularen Modellverbindung zur

photophysikalischen Charakterisierung vorgestellt. Die BODIPY-Verbindungen wurden durch

Kernspinresonanzspektroskopie (NMR), Massenspektrometrie (MS), Infrarotspektroskopie

(FT-IR), UV-Vis-Messungen sowie Röntgenstrukturanalyse charakterisiert. Die

BODIPY-beinhaltenden Polymere wurden weiteres durch Gelpermeationschromatographie (GPC)

analysiert und mittels Kalibrierung auf dem UV-Vis-Spektrometer quantifiziert. Die Freisetzung der

gekoppelten Substanz durch die Bestrahlung mit Licht wurde ebenfalls mittels UV-vis Messungen

gezeigt.

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VI

Table of contents

Acknowledgement ..................................................................................................................... III

Abstract .................................................................................................................................... IV

Zusammenfassung .................................................................................................................... V

Table of contents ...................................................................................................................... VI

1 Introduction ......................................................................................................................... 1

1.1 Stimuli-responsive polymers ......................................................................................... 1

1.1.1 Temperature- / pH-responsive polymers ................................................................ 1

1.1.2 Photo-responsive polymers ................................................................................... 2

1.2 Stimuli-responsive drug delivery systems ..................................................................... 2

1.2.1 Hydrogels / Microgels ............................................................................................ 4

1.2.2 Self-assembled structures ..................................................................................... 4

1.2.3 Photocleavable protecting groups .......................................................................... 4

1.3 BODIPY ........................................................................................................................ 7

1.4 Poly(organo)phosphazenes ........................................................................................ 12

1.5 Click chemistry ........................................................................................................... 14

1.5.1 Copper catalyzed azide-alkyne cycloaddition (CuAAC) ....................................... 14

2 Aim of the thesis ............................................................................................................... 16

3 Experimental ..................................................................................................................... 17

3.1 Materials, methods and devices .................................................................................. 17

3.2 BODIPY synthesis and modification ........................................................................... 20

3.2.1 Synthesis of BODIPY framework ......................................................................... 20

3.2.2 Synthesis of hydrolysed BODIPY ......................................................................... 21

3.2.3 Synthesis of BODIPY-azide via bromination and azidation .................................. 21

3.3 Poly(organo)phosphazene synthesis .......................................................................... 22

3.3.1 Synthesis of the precursor poly(dichloro)phosphazene ........................................ 22

3.3.2 Polymer 1: Mixed macrosubstitution .................................................................... 22

3.3.3 Polymer 2: Macrosubstitution ............................................................................... 23

3.3.4 Polymer 3: BODIPY-containing macrosubstitution ............................................... 24

3.4 Synthesis of poly(ethylene glycol) monomethyl ether azide ........................................ 24

3.4.1 Tosylation of poly(ethylene glycol) monomethyl ether .......................................... 24

3.4.2 Azidation of PEG ................................................................................................. 25

3.5 Amine protection of propargylamine ........................................................................... 25

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VII

3.5.1 BOC-protection of propargylamine ....................................................................... 25

3.5.2 Fmoc-protection of propargylamine ..................................................................... 26

3.6 Copper-catalyzed azide–alkyne cycloaddition (CuAAC): small molecules .................. 27

3.6.1 General procedure ............................................................................................... 27

3.6.2 Synthesis of pentyne-BODIPY ............................................................................. 27

3.6.3 Synthesis of BOC-propargylamine BODIPY ........................................................ 28

3.6.4 Synthesis of Fmoc-propargylamine BODIPY ....................................................... 28

3.6.5 Synthesis of 3-ethynylaniline-BODIPY ................................................................. 28

3.7 Copper-catalyzed azide–alkyne cycloaddition (CuAAC): macromolecules .................. 29

3.7.1 General procedure ............................................................................................... 29

3.7.2 2.7 w% BODIPY-azide on Polymer 1 ................................................................... 30

3.7.3 9.6 w% BODIPY-azide on Polymer 1 ................................................................... 30

3.7.4 PEG-azide on Polymer 2 ..................................................................................... 30

3.7.5 BODIPY-azide on 25 % PEG-substituted Polymer 2 ............................................ 30

3.8 Coupling methods testing ........................................................................................... 31

3.8.1 Reaction of the hydrolysed BODIPY with phenoxyacetyl chloride ........................ 31

3.8.2 Reaction of the hydrolysed BODIPY with benzoic acid ........................................ 32

3.9 Model compound: reaction of the photoreactive OH-group ......................................... 32

3.9.1 Reaction of pentyne-BODIPY with phenoxyacetyl chloride .................................. 32

3.9.2 Reaction of pentyne-BODIPY with phenylacetic acid ........................................... 33

3.10 Reaction of BODIPY-azide with phenylacetic acid ...................................................... 34

3.11 Loading on polymer .................................................................................................... 34

3.11.1 CuAAC of phenylacetic acid-BODIPY-azide with Polymer 1 ................................ 34

3.11.2 Loading of phenylacetic acid on Polymer 1 bearing 9.6 w% BODIPY .................. 34

4 Results and discussion ..................................................................................................... 36

4.1 BODIPY synthesis and modification ........................................................................... 36

4.1.1 Synthesis of the BODIPY framework ................................................................... 36

4.1.2 Synthesis of the hydrolysed BODIPY ................................................................... 39

4.1.3 Synthesis of BODIPY-azide ................................................................................. 41

4.2 Poly(organo)phosphazene synthesis .......................................................................... 46

4.2.1 Synthesis of the precursor poly(dichloro)phosphazene ........................................ 46

4.2.2 Polymer 1: mixed macrosubstitution .................................................................... 46

4.2.3 Polymer 2: macrosubstitution ............................................................................... 47

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VIII

4.2.4 Polymer 3: BODIPY-containing macrosubstitution ............................................... 48

4.3 Synthesis of poly(ethylene glycol) monomethyl ether azide ........................................ 53

4.4 Copper-catalyzed azide-alkyne cycloaddition: small molecules .................................. 54

4.4.1 Synthesis of pentyne-BODIPY ............................................................................. 55

4.4.2 Synthesis of BOC-/Fmoc-propargylamine-BODIPY and tested deprotection

methods ............................................................................................................................ 56

4.4.3 Synthesis of 3-ethynylaniline-BODIPY ................................................................. 58

4.5 Copper-catalyzed azide-alkyne cycloaddition: macromolecules .................................. 58

4.5.1 BODIPY-azide on Polymer 1 ............................................................................... 58

4.5.2 PEG-azide on Polymer 2 ..................................................................................... 66

4.5.3 Coupling of BODIPY-azide on 25 % PEG-substituted Polymer 2 ......................... 68

4.6 Coupling methods testing ........................................................................................... 70

4.7 Model compound: reaction of the photoreactive OH-group ......................................... 71

4.8 Reaction of BODIPY-azide with phenylacetic acid ...................................................... 74

4.9 Loading on polymer .................................................................................................... 75

4.9.1 Coupling of 9.6 w% BODIPY on Polymer 1 with phenylacetic acid ...................... 75

4.9.2 CuAAC of phenylacetic acid-BODIPY-azide with Polymer 1 ................................ 75

4.10 Comparison of BODIPY-Polymer coupling methods ................................................... 77

5 Conclusion and outlook ..................................................................................................... 79

References ............................................................................................................................... 81

Appendix ................................................................................................................................... 88

Pauline Stadler

1

1 Introduction

1.1 Stimuli-responsive polymers

Stimuli-responsive polymers are generating considerable interest nowadays due to their manifold

possible fields of application. In the last years, researchers have been focused on biomedical

uses, for example drug delivery systems 1, tissue generation and repair 2, biosensors 3, smart

coatings 4 or artificial muscles 5. The main characteristics of stimuli-responsive polymers that

make them a remarkable tool for those fields are their variations of behavior in response to

environmental changes 6. Hence, exposure to physical or chemical stimuli, such as temperature 7,

pH 8, light 9, mechanical force 10, redox 11, ionic strength 12 or bioactive species 13 will induce a

change of properties. This behavior can be considered to be biomimetic, since the response to

the environment is also used for the function of living cells 14. Stimuli-responsive characteristics

are generated by functional groups that are incorporated or attached to a polymer chain.

Depending on the functionality, external stimuli can lead to conformational or phase changes, as

well as other responses. A new field of research are multi-stimuli responsive polymers, where a

combination of functional groups within one polymer results in respond to different stimuli 15.

1.1.1 Temperature- / pH-responsive polymers

The most well-documented and understood stimulus so far is temperature. A fundamental

phenomenon is the lower critical solution temperature (LCST) 16, which is defined by the critical

temperature, below which the components of a mixture are miscible. Above the LCST, phase

separation occurs due to an entropically driven process. This process is a temperature-triggered

hydrophobic-hydrophilic transition that is fully reversible. Hence, cooling below the LCST leads to

regeneration of the initial state of the polymer 15. This behavior is traditionally observed in polar

media, such as water and alcohols, due to its dependency on hydrogen-bond capabilities 6,16. An

extensively studied polymer is poly(N-isopropylacrylamide) (PNIPAm) because it has an LCST of

about 32 °C, which is close to body temperature, making it attractive for biomedical

applications 6,14,17. Above the LCST PNIPAm undergoes a transition from a random coil, which is

enthalpically favored, to a dense globular, entropically favored structure 15. Copolymerization with

hydrophilic or hydrophobic monomers enables tuning of the LCST to higher or lower temperatures.

Moreover, the LCST is not only dependent on the chemical composition, but also on parameters,

such as chain length 18,19, tacticity 20,21, pressure 22,23 and chemical nature of the end-group 24,25.

For completion, the existence of an upper critical solution temperature (UCST) should be

mentioned as well, which describes an opposite behavior compared to the LCST 26.

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Polymers that respond to pH-changes can be made through incorporation of ionizable monomers

into the polymer backbone 6,14. Functional groups, like carboxylic acids, can donate or accept

protons, which allow phase transitions and solubility changes, depending on the environmental

pH-value. The pH influences ionic interactions that can lead to extension or collapsing of the

polymer, due to electrostatic attraction or repulsion of charges 8,15. A common example for this

behavior is poly(acrylic acid), that swells under basic conditions 27.

Disadvantages of temperature or pH responsive polymers are that a very precise control over

polymer synthesis is necessary to obtain defined molecular weight, architecture, monomer content

and block distribution. Temperature responsive polymers also suffer from the drawback that local

temperature changes are hard to control in medical applications 14.

1.1.2 Photo-responsive polymers

Light as a stimulus is attracting widespread interest due to its controllable parameters in terms of

irradiation time, wavelength, space and light intensity 28–30. Photo-responsive moieties that are

introduced into a polymer chain are responsible for the light-triggered property change. Depending

on the used compound, a specific wavelength has to be applied, to get the desired behaviour. An

advantage of light over other stimuli is that it can be externally applied with high accuracy at a

defined area or volume 31. Due to the broad spectrum of the light, a variety of systems can be

chosen, reaching from the near infrared light to the hard UV-light 15. This allows application of

numerous photoactive functionalities that can be triggered through different wavelengths. Two

main photo-induced responses are commonly considered, where isomerization between two or

more states is one of them. The second approach is the usage of photocleavable protecting

groups, where a caged molecule can be released through irradiation. Depending on the moiety

and its response to light, the reaction can be reversible, as it is known for azobenzenes 32,33, or

irreversible, like for o-nitrobenzyl esters 30,31.

1.2 Stimuli-responsive drug delivery systems

Drug delivery systems have become a central role in biomedicine to overcome certain issues of

common medication types. The principal characteristics of drug delivery systems are that they

release the active substance at the desired region in the right concentration. With this method,

side effects through uncontrolled biodistribution should be minimized 9. In general, an ideal drug

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3

delivery system should be simply administered, able to deliver the cargo to desired locations and

composed of non-toxic, biocompatible and biodegradable components 6. Other prerequisites,

such as high stability, increase of drug circulation time and accumulation near the target, should

also be fulfilled. Those challenging requirements are responsible for the application of stimuli-

responsive polymers as drug delivery systems 6,9. In Figure 1 are three examples for controlled

drug delivery systems depicted, where the drug release is shown through the shrinkage of a

gel (a), destruction of micelles (b) and a light triggered activation of a photoprotecting group (c).

The advantages and disadvantages of those approaches are further described in the following

chapters.

Figure 1: Examples of drug delivery systems: a) gels, b) micelles, c) photoprotecting groups (PPGs).

Pauline Stadler

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1.2.1 Hydrogels / Microgels

One of the main strategies for controlled drug delivery is the usage of highly crosslinked polymers,

such as hydrogels or microgels 6,34. Their stimuli-response mostly results in reversible collapse

and expansion of the gel. Porosity and water-swellability enable drug loading into the gel matrix.

A subsequent release occurs due to gel collapse, triggered by external stimuli, like the

temperature. This process is dependent on the diffusion rate of the drug-molecule, as well as the

applied stimulus 14. Examples of this behaviour are PNIPAm-hydrogels 35, which keep a drug in its

matrix in its swollen state. At temperatures above the gel collapse point, a rapid release of

captured water and co-solutes, like the therapeutic agent, takes place. After this initial burst

release, a slower diffusion of the target molecule from the shrunken gel takes place 14. Other

stimuli, such as light-triggered heating 36 or release, are commonly discussed as well.

Consequently, hydrogels and microgels are promising drug delivery systems, especially if

biodegradability of the gel is enabled. In comparison to hydrogels, microgels exhibit several

benefits, as their response rate is faster and long circulation in blood streams are possible 37.

Despite its advantages, the applications of crosslinked gels have been limited due to their lack of

control in terms of drug-release and gel-synthesis. Moreover the response times may be too long

for the majority of therapeutic uses 6,14.

1.2.2 Self-assembled structures

Another widely considered stimuli-responsive polymer architectures for drug delivery are self-

assembled structures, such as micelles. They commonly consist of block co-polymers with two

thermodynamically incompatible segments, which are responsible for the self-organization in

solution or bulk 15,38. Their fragile architecture is leading to a rapid response by stimuli that disrupt

the self-assembling. Thus, therapeutic agents, which are commonly of a hydrophobic nature, can

be encapsulated in the hydrophobic core of the micelle and released by interruption of the

hydrophobic / hydrophilic interactions 6,15. With this drug delivery method, independence of the

diffusion rate of the cargo is enabled, as well as fast response 15.

1.2.3 Photocleavable protecting groups

Photocleavable protecting groups (PPG) are becoming increasingly important as drug delivery

systems, since light as a stimulus has some outstanding advantages 9. Their ability to cage and

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deactivate a substrate through covalent bonds and subsequently release and reactivate them

upon irradiation 39 makes them a fascinating tool for many applications, also including organic

synthesis 40 and material science 41. A basic requirement for photocages is, besides a high

quantum yield of release, a narrow and distinct absorption maximum 31. Outside this absorbance

maximum range, the absorption should be low. Further sought-after properties are chemical

stability, solubility in an aqueous environment and low toxicity 42. Although there is a broad light

spectrum, only a small range of wavelengths can be used for therapeutic applications, known as

the phototherapeutic window. UV-light in the range of 254 - 400 nm is the major trigger for most

PPGs, causing most photoreactions due to its high energy 9. Objectively, it can rarely be applied

on humans, since it is harmful to cells and may lead to skin cancer and cell injury through molecular

damage of lipids, nucleic acids and proteins 43. Moreover, the application of UV-light is limited

through its small penetration depth in human tissue, depicted in Figure 2 9,44. With this major issue,

drug delivery is unfeasible, as internal organs would be not reachable. Thus, the wavelength for

deprotection should be as high as possible to reach the therapeutic window at wavelengths of

λ = 650 – 900 nm 42. Visible light and near-infrared light (NIR) are attractive alternatives to UV-

light, but suffer from the drawback that less photochemical reactions are possible due to the low

energetic state 9.

Figure 2: Scheme of tissue penetration depth at several wavelengths (adjusted from literature 9).

5

4

3

2

1

0

Tis

su

e p

en

etr

atio

n d

ep

th /

mm

wavelength / nmUV Purple Blue Green Yellow Orange Red NIR

380 450 495 570 590 620 750

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Well-known examples of photocages are o-nitrobenzyl (ONB) 45,46, phenacyl 47 , coumarin 48 and

benzyl-based 49 derivatives, whose structures and deprotection can be seen in Figure 3. Most

PPGs are based on unsaturated systems consisting of aromatics, nitro and carbonyl groups.

Preliminary ONB (Figure 3 a) and its deprotection has been widely studied, where it was found

that upon irradiation an excited state is formed, followed by an intramolecular hydrogen abstraction

and subsequent ring-opening, leading to a release of the leaving group and

2-nitrosobenzaldehyde as byproduct 45. Numerous leaving groups can be cleaved from the ONB

PPG, such as phosphates 50, carboxylates 51, carbonates 50, carbamates 52, alkoxides 50, etc.

Commonly, wavelengths between λ = 345 – 420 nm are necessary for the deprotection 40,41,53, but

modifications can shift the absorbance to longer wavelengths. Therefore electron-withdrawing

groups (EWGs) in the para-position and electron-donation groups (EDGs) in the meta-position

were used to obtain this effect 31. The nitrosobenzaldehyde photo-byproduct of the ONB is

considered to be problematic, since it may decrease the efficiency of releasing an amine and can

interfere with biological systems. In contrast to this, the photo-byproduct of phenacyl (Figure 3 b)

is chemically and photochemically stable. The phenacyl deprotection mechanism and efficiency

is strongly dependent on its substitution on the phenacyl structure, as well as leaving group and

reaction conditions, especially the chosen solvent. Closely related to the phenacyl are coumarin

PPGs (Figure 3 c) that show some structural similarities. Coumarin PPGs are increasingly

becoming more important for biological and biomedical applications due to their fast release of a

good leaving group. Another highly modifiable class of PPGs are benzene-based derivatives, like

pyrene 1-yl methyl PPG (Figure 3 d). Structural alterations of those photocages can be classified

into three categories: substitution on the benzene ring, expansion of the aromatic system and

modification at the benzylic carbon 45. Numerous other photocages are nowadays known, but most

of them suffer from the major issue, that they only operate at UV-light, which limits the application

as drug delivery systems. Hence, photocleavable protecting groups that absorb in the visible light

spectrum are needed. Recent developments regarding new visible-light absorbing photocages

have led to the investigation of meso-substituted boron-dipyrromethene (BODIPY) derivatives,

where the main focus of this thesis will be 42,54.

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Figure 3: Different types of photoprotecting groups (PPGs) and their deprotection. LG = leaving group;

a) ortho-nitrobenzyl PPG (ONB), b) phenacyl PPG, c) coumarin 4-yl methyl PPG, d) pyrene 1-yl methyl

PPG; adapted from literature 30,45.

1.3 BODIPY

Difluoroboradiaza-s-indacenes, generally termed boron-dipyrromethenes (BODIPYs) 55, are

gaining widespread interest due to their diverse areas of application. They have been widely

studied as laser dyes 56, photocages 42, photosensitizers 57, sensors 58, catalysts 59, fluorescent

indicators 60, probes for bioimaging 61 and components of solar cells 62. This is caused by their

outstanding properties, including high fluorescence quantum yields, high molar absorption

coefficients, chemical and photochemical stability and low toxicity 42,55,63. Moreover, their optical

properties can be easily adapted by modifications of the BODIPY-core. For example, as illustrated

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in Figure 4, the slight structural change of the BODIPY-framework to the azaBODIPY-framework,

specifically the change of one CH2 moiety to a nitrogen atom, results in a distinct bathochromic

absorbance shift of about 100 nm. Notably, due to the instability of those displayed simple

framework structures, at least one substituent is introduced at the pyrrole moieties 64.

Figure 4: Chemical structures of BODIPY and azaBODIPY-framework with IUPAC numbering of the

carbons, adapted from literature 64.

Recent developments have led to the investigation of novel meso-methyl BODIPYs as excellent

photocages, which can release a compound upon irradiation with light in the visible up to the NIR

light spectrum 65. The photorelease mechanism, schematically shown in Figure 5, most likely

occurs due to a photochemical SN1 reaction via a carbocation intermediate and subsequent

substitution through nucleophilic attack of the solvent 54. As described above, BODIPYs show ideal

characteristics as photocleavable protecting groups for biomedical applications, as they are

outstanding chromophores with tunable absorption wavelengths, have no chiral center and are

stable in the dark 66. Major issues of known meso-methyl BODIPY compounds, however, are their

low quantum yields of photorelease. Through structure-activity relationship studies the correlation

between the photochemical reactivity and the variations of the core- and boron-substituents, as

well as the nature of the leaving group, were established, improving in this aspect and providing

BODIPY photocages with increased quantum yields of photorelease 54.

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Figure 5: Mechanism of photorelease of a leaving group (LG) from a meso-methyl BODIPY photocage via

a carbocation intermediate and subsequent reaction with methanol, adapted from literature 54.

Different sites for modification and hence property-tuning are possible. They reach from the pyrrole

carbons at position 1, 2, 3, 5, 6 and 7, over the central 8- or meso-position, to the boron centrum

at position 4 55,63. Recently, it was found that the C2 and C6 positions have the highest negative

partial charge, making them most reactive for electrophilic aromatic substitutions 64. Based on

such reactions, the di-halogenated BODIPY is the most investigated compound, where a

red-shifted absorption maximum (544 – 556 nm) in comparison to the non-halogenated substance

(529 - 534 nm) arises 54. A correlation between the intersystem crossing (ISC) efficiency and the

atomic number of the substituents was found (H < Cl < Br < I), caused by the introduction of heavy

atoms. Hence, the quantum yields of photorelease of BODIPY-photocages are improved by

increasing the molecular weight of the 2,6-substituents. The Jablońsky-diagram in Figure 6

explains the high quantum yields of photorelease for halogenated BODIPYs with the fact that the

release of the leaving group must happen at least partially from the triplet excited state 54. Beside

these 2,6-modifications, methyl substituents on position 1, 3, 5 and 7 are acidic enough to undergo

a Knoevenagel condensation reaction with activated aromatic aldehydes to extend the π-system,

leading to a bathochromic absorbance shift, as well 64. Boron alkylation could extremely improve

the quantum yields of photorelease, while alkylation on the meso-methyl position showed no

effect 54.

Beside modifications of the BODIPY-framework, also the leaving group quality affects the

efficiency and the quantum yield of photorelease. This effect is nowadays well-known and studied

for other photocleavable protecting groups 45. The quality of the leaving groups is correlated to

their pKA-value of their conjugated acids, where lower values can be assigned to better leaving

groups. Examples for poor leaving groups are amines, alcohols and thiols, which have to be linked

through different groups, like carbamates or carbonates 54,67.

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Figure 6: Jablońsky diagram of a 2,6-substituted Cl-BODIPY compound, adapted with permission from 54,

Copyright © 2017, American Chemical Society.

The introduction of substituents can be done via two different approaches: pre-functionalization

and post-functionalization, where modifications are done before or after the framework formation,

respectively. Examples of both methods are shown in Figure 7, where for the pre-functionalization

method, substituted pyrroles are used as starting materials 64,68,69. According to the

pre-functionalization route, symmetric BODIPYs can be obtained by the reaction with aldehydes,

which is commonly done via an acid-catalyzed condensation and subsequent oxidation, followed

by complexation with boron trifluoride 68. For the synthesis of asymmetric BODIPYs, 2-acylpyrrole

can be used instead of the aldehyde 69. Numerous other starting materials and reactions are

possible, allowing an easy access to a high diversity of BODIPY-structures 64,68,69. Despite this

fact, some modifications are still challenging using the pre-functionalization approach. Hence,

post-functionalization offers many other possibilities for the introduction of substituents, like

electrophilic or nucleophilic substitution, Knoevenagel condensation and Suzuki coupling 63.

However, the method of choice depends on the structure of the target molecule and the efficiency

of the possible synthesis techniques.

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Figure 7: Examples of functionalization methods of BODIPY compounds according to literature 64.

A) Prefunctionalization, B) Postfunctionalization.

While BODIPYs have outstanding properties for the usage in biomedical applications, they are

rather limited by their hydrophobicity. Since water-solubility is essential for the utilization as drug

delivery systems, several approaches were investigated to overcome this drawback 70–72.

Traditionally, water-solubility is achieved with the installation of ionizable hydrophilic groups 73–75,

such as carboxylic acids, phosphonic acids, sulfonic acids and ammonium groups or by

attachment to a hydrophilic polymer 76–78, like poly(ethylene glycol) or carbohydrates. Current

studies by Weinstein and others have been focused on the introduction of sulfonate groups, where

first the electrophilic substitution at the 2,6-positions with chlorosulfonic acid was the preferred

synthesis method. The product, shown in Figure 8 a), could be obtained in good yields and water-

solubility. However, chlorosulfonic acid is highly reactive, which can lead to product mixtures due

to the labile BODIPY-core and purification is quite difficult for such compounds 70,71. Moreover, it

was found that 2,6-sulfonation of meso-methyl-BODIPY photocages is incompatible with their

photoreaction, since the excited state barrier of the photorelease is increased 72. Hence, another

sulfonation technique was investigated were 2-mercaptoethane sulfonium acid sodium salt was

attached to the 3,5-position via nucleophilic substitution of a bromine moiety, which can be

introduced with the usage of N-bromosuccinimide (NBS). The resulting product, depicted in

Figure 8 b), exhibits improved water-solubility, high photorelease efficiency and control over

cellular location via the degree of sulfonation 72. Another reported method to increase the

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hydrophilicity includes attachment of oligo(ethylene glycol) moieties, which also enables cell

permeability and tumor targeting characteristics 79. Generally, only few methods are currently

known to improve water-solubility of BODIPY-compounds and retain their photoprotecting group

properties.

Figure 8: Water soluble BODIPY-structures synthesized via: a) sulfonation at the 2,6-position 71,

b) sulfonation via electrophilic substitution with 2-mercaptoethane sulfonium acid sodium salt at the

3,5-position 72.

1.4 Poly(organo)phosphazenes

Polyphosphazenes, illustrated in Figure 9, are polymers consisting of an inorganic backbone with

alternating phosphorus and nitrogen atoms, substituted with two side groups on the phosphorus.

If the substituents are of organic nature, the resulting polymers are called

poly(organo)phosphazenes and commonly synthesized starting from the high reactive precursor

poly(dichloro)phosphazene 80. Traditionally, the precursor can be obtained by thermal ring-

opening polymerisation or living cationic polymerisation 81, followed by a process called

macromolecular substitution, where the chlorine moieties were substituted, resulting in

poly(organo)phosphazenes. This post-polymerisation process has to be carried out under

anhydrous conditions, due to the high reactivity of the precursor that can be rapidly hydrolysed.

Using different organic substituents, commonly primary amines and alcohols, within one polymer

allows for a broad variety of possible structures and properties 80. Hence,

poly(organo)phosphazenes are promising materials for various fields of application like vaccine

delivery 82, tissue engineering 83 and fuel cell membranes 84. Other obtainable characteristics,

such as biocompatibility and backbone degradability especially make them interesting for

biomedical uses 80,85.

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Figure 9: General structure of the macromolecular precursor poly(dichloro)phosphazene and the final

poly(organo)phosphazene, with R = organic substituents 80.

The degradation of poly(organo)phosphazenes, schematically shown in Figure 10, occurs via

hydrolysis of the backbone after substitution of the organic substituents. This leads to the fragile

hydroxyphosphazene and phosphazane. As a result of cleavage of either the backbone or the

substituents, further degradation leads to phosphates and ammonium salts as final degradation

products, which are reported to be benign. Depending on the attached organic substituent, it is

possible to obtain completely non-toxic degradation products 80,86. Previous research showed that

a hydrophilic Jeffamine M-1000 substituent is considered to enable excellent water-solubility of

the polymer, as well as possible degradation. Additionally, its degradation products and

intermediates make these polymers biocompatible and therefore promising for biomedical

applications 85.

Figure 10: Proposed mechanism for the hydrolytic degradation of a poly(organo)phosphazene 80.

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1.5 Click chemistry

The widely known concept of click chemistry describes reactions that are easy to perform, serve

high product yields with little or no byproducts, have a high functional group tolerance and can be

carried out well under many conditions 87. The simplicity of these reactions gives access to

numerous possible modifications that are of particular interest for many application fields,

especially polymer chemistry. Representative examples for click reactions are azide-alkyne

cycloadditions 88, thiol-ene/yne reactions 89, Diels-Alder reactions 90 and 1,3-dipolar

cycloadditions 91, where in this thesis only the azide-alkyne cycloaddition will be discussed in more

detail 92.

1.5.1 Copper catalyzed azide-alkyne cycloaddition (CuAAC)

The copper-catalyzed azide-alkyne cycloaddition (CuAAC) is a common investigated type of click

reaction. In this reaction, organic azides and terminal alkynes form 1,4-disubstituted 1,2,3-triazoles

through a catalytic cycle, shown in Figure 11. A Cu(I)-catalyst is required for fast kinetics at room

temperature and regioselectivity, while in the thermal process mixtures of regioisomers are

obtained 93. Generally, the reaction tolerates protic and aprotic solvents, as well as most functional

groups. The formed triazole ring possess remarkable features such as high chemical stability,

strong dipole moment, aromatic character and hydrogen bond acceptor property 87. Despite those

striking characteristics of the reaction, the CuAAC suffers from some major drawbacks. Common

issues are the instability of organic azides that make them hard to handle, the contamination of

the product by the catalyst and the instability of the Cu(I)-oxidation state, which can, however, be

solved by the addition of a reducing agent, like sodium ascorbate 94.

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Figure 11: Proposed catalytic cycle for CuAAC, adapted from literature 95.

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2 Aim of the thesis

This thesis represents a new approach towards green-light photo-responsive drug delivery

systems, combining meso-methyl-BODIPY derivatives as photocleavable protecting groups and

poly(organo)phosphazenes as carrier. The aim of this work includes synthesis of a water-soluble

polymer with attached BODIPY moieties acting as drug delivery system for a cargo. Different

substituents on the poly(organo)phosphazene were chosen to achieve the desired properties of

the polymer, namely Jeffamine or poly(ethylene glycol) for water-solubility and propargylamine as

reactive moiety for further functionalization. Phenylacetic acid and phenoxyacetyl chloride were

used as model-cargo compounds. For the reaction of the BODIPY-compound with the polymer

click chemistry, in detail copper-catalyzed azide-alkyne cycloaddition, was performed due to its

reported robust synthesis and adaptability. Another tested approach was to directly use an amine-

functionalized BODIPY-compound during the synthesis of the polymer as a macrosubstituent.

Finally, this work also includes the synthesis of a small model compound for photophysical

characterization and photorelease studies.

A general structure and concept of the photorelease of the desired compound is demonstrated in

Figure 12.

Figure 12: Schematic principle of the aims of the work presented in this thesis.

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3 Experimental

3.1 Materials, methods and devices

Nuclear magnetic resonance (NMR) spectra were recorded on a Bruker® Avance 300

spectrometer (300 MHz). All spectra were measured at room temperature in deuterated

chloroform. The signals were reported in response to their solvent signal at 7.23 ppm. The

multiplicities of the signals are denoted by s (singlet), d (doublet), t (triplet), q (quartet) and m

(multiplet). 13C-shifts of correlated 13C and 1H were obtained from HSQC-measurements. Infrared

spectra were measured on a PerkinElmer Spectrum 100 FT-IR Spectrometer. UV-vis spectra were

recorded on a SpectraMax® M2e Mulit-Mode Microplate Reader with applied wavelengths of

350 - 780 nm. For lower wavelengths down to 190 nm a PerkinElmer Lambda 25 UV/VIS

Spectrometer was used. For GPC measurements a Viscothek GPCmax® with three GRAM-

columns (1 x 30 Ǻ, 2 x 100 Ǻ; 300 x 8 mm, 10 µm particle size), purchased from PSS, was used.

The samples were filtered through 0.2 µm PTFE syringe filters prior to injection and eluted with

DMF containing 10 mM LiBr as the mobile phase at a flow rate of 0.75 ml min-1 at 60 °C. The

samples were detected via a light scattering (LS), refractive index (RI) and viscosity detector and

molecular weights were estimated by a conventional calibration of the RI detector against linear

polystyrene standards from PSS. Water-soluble products were measured on an Agilent

Technologies 1260 Infinity II instrument equipped with Shodex OHpak LB-802.5 (300 x 8 mm,

5 µm particle size) and LB-804 (300 x 8 mm, 5 µm particle size) columns, detected with a UV-vis

detector from Agilent, a refractive index detector RI-501 from Shodex and a TREOS II light

scattering detector from Wyatt technology. Samples were filtered through 0.2 µm nylon syringe

filters prior to injection. As an eluent Milli-Q-water with 0.1 M NaNO3 and 0.0251 w% NaN3 at a

flow rate of 0.5 mL min-1 was used. BODIPY compounds were hereby detected with the UV-vis

detector at an adjusted wavelength of 540 nm. High resolution mass spectra were recorded on an

Agilent 6520 ESI-QTOF (Agilent Technologies, Waldbronn, Germany) in positive mode with

methanol as an eluent. Thin layer chromatography was performed on pre-coated TLC sheets

ALUGRAM® SIL G/UV254 with 0.20 mm silica gel 60, purchased from Machery-Nagel. For

detection UV-light with a wavelength of 254 nm or 365 nm was used. Water was removed by an

Alpha 1-2 LDplus freeze drying system from Christ. Degradation studies were performed on a

365 nm photochemical reactor. Dialysis tubes were purchased from Spectrum Laboratories, Inc.

For ultrafiltration Vivaspin 20 tubes (3 kDa MWCO) from GE Heathcare were used.

Photorelease studies of the model compound were carried out at the Department of Chemistry at

Masaryk University, Brno, Czech republic.


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Suitable single crystals for X-ray diffraction were obtained under ambient conditions from a

solution of BODIPY in n-heptane / ethyl acetate by evaporation of the solvent. Diffraction data

were collected on a Bruker Smart Apex diffractometer operating with MoKα radiation (λ = 0.71073

Å). The structures were solved by direct methods (SHELXL-2014/7) and refined by full-matrix least

squares on F2 (SHELXL-2014/7) [Sheldrick GM (2015) Acta Cryst C 71:3]. The H atoms were

calculated geometrically, and a riding model was applied in the refinement process.

All used chemicals and solvents are alphabetically listed in Table 1. Ethanol for dialysis was

purchased from VWR and redistilled after usage. Triethylamine from Merk was distilled and dried

over molecular sieves (3 Ǻ) bevor usage. Anhydrous dichloromethane was purified through a

solvent purification column (MBraun SPS compact). Chloroform-d for NMR-measurements was

dried over molecular sieves (3 Ǻ). Milli-Q-water was obtained from a Millipak® Express 40 with a

0.22 µm filter. All other reagents and solvents were purchased and used as received without any

additional purification. Water-sensitive and highly reactive chemicals were stored in the glovebox

under an inert atmosphere. All BODIPY-compounds were stored in the absence of light at room

temperature, to avoid photodegradation.

Table 1: Overview of used chemicals.

Chemical Provider Purity

(+)-sodium-L-ascorbate Sigma Aldrich 99,00%

(trimethylsilyl)phosphorimidoyl trichloride (monomer) Synthesized in house -

2-chloro-2-oxoethyl acetate (acetoxyacetyl chloride) Alfa Aesar & Acros

Organics 97.00%

3-ethyl-2,4-dimethyl-1H-pyrrole (kryptopyrrole) Alfa Aesar & Sigma

Aldrich

96%,

97%

3-ethynylaniline TCI >98.0%

4-dimethylaminopyridine (DMAP) Sigma Aldrich ≥99%

Boron trifluoride diethyl etherate Alfa Aesar >98%

Chloroform VWR 99.0%

Chloroform-d Eurisotop 99,80%

Copper sulfate pentahydrate J.T. Baker 98,50%

Dichloromethane (DMC) Chem-Lab 99.8+%

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Dichloromethane anhydrous (DCM) Alfa Aesar 99.7%

Dichlorotriphenylphosphoran Sigma Aldrich 95,00%

Di-tert-butyl dicarbonate Fluorochem -

Ethanol VWR 99%

Ethanol, absolute for analysis Chem-Lab for

analysis

Ethyl acetate VWR 99,90%

N-(9H-fluoren-9-ylmethoxycarbonyloxy)-succinimid

(Fmoc-Osu) Fluorochem

Hydrochloric acid VWR 37.0%

Jeffamine M-1000 Huntsman -

Kieselguhr - 325 Mesh powder (celite) Alfa Aesar -

Magnesium sulfate VWR -

Methanol VWR >99%

N,N′-dicyclohexylcarbodiimide (DCC), Sigma Aldrich 99,00%

N,N-diisopropylethylamin (DIPEA) TCI >98.0%

N,N-Dimethylformamide (DMF) VWR >99.9%

N-bromosuccinimide (NBS) Fluorochem -

n-heptane VWR 99,90%

Pentyne TCI >98.0%

Phenoxyacetyl chloride Alfa Aesar 98,00%

Phenylacetic acid Sigma Aldrich 99,00%

Poly(ethylene glycol) monomethyl ether, 550 Alfa Aesar -

Propargylamine Fluorochem -

p-toluenesulfonyl chloride Acros Organics ≥99%

Pyridine Roth >99.0%

Silica gel 60 (0.015-0.040 mm) Merck -

Sodium azide Sigma Aldrich >99.5%

Sodium chloride VWR 98,00%

Sodium hydrogen carbonate VWR -

Sodium hydroxide J.T. Baker 98,00%

Tetrahydrofuran anhydrous Alfa Aesar 99,80%

Triethylamine Merk dest.

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3.2 BODIPY synthesis and modification

3.2.1 Synthesis of BODIPY framework

The BODIPY framework was synthesized according to slightly adjusted literature procedure 42,

using kryptopyrrole and acetoxyacetyl chloride as starting materials.

1.0 mL of kryptopyrrole (2.2 eq. 7.1 mmol) was dissolved in 40 mL dry DCM and the solution was

put under argon atmosphere. Afterwards, 0.36 mL acetoxyacetyl chloride (1 eq., 3.2 mmol) were

added dropwise through a septum upon which the color changed from yellow to red. The reaction

mixture was allowed to stir for 24 hours in the dark at room temperature. After this time a first

batch of 2.7 mL triethylamine (6 eq., 19.3 mmol) was added dropwise to the solution and the

reaction mixture was stirred for 15 minutes. Then 3.7 mL of anhydrous boron trifluoride diethyl

etherate (9 eq., 29.0 mmol) were added slowly and the solution was stirred for 1 hour. A second

batch of 2.7 mL triethylamine was added and stirred again for 15 minutes. Then again, 3.7 mL of

boron trifluoride diethyl etherate were added and stirred for 1 hour. Afterwards, DCM was

evaporated and the product was re-dissolved in 100 mL of ethyl acetate. The organic layer was

washed with brine (3 x 80 mL) and the aqueous phases were combined and re-extracted with

50 mL of ethyl acetate. The organic phases were dried over magnesium sulphate and the solvent

was then evaporated under reduced pressure with celite to dryness. The product was purified by

dry column vacuum chromatography (DCVC) with 30 mL fractions of n-heptane and ethyl acetate

as mobile phase with a gradient of 1 mL increase of ethyl acetate every second fraction, starting

with pure n-heptane. The product was obtained as a dark red and golden precipitate with a yield

up to 40 % (485 mg).

1H-NMR (300 MHz, CDCl3): δ = 1.03-1.08 ppm (t, 3 H, -CH2-CH3), 2.15 ppm (s, 3 H, C-O-CH3),

2.27 ppm (s, 6 H, -CH3), 2.36-2.41 ppm (q, 4 H, -CH2-CH3), 2.52 ppm (s, 6 H, -CH3), 5.33 ppm

(s, 2 H, CH2-O-Ac) [Appendix 1]

λmax = 544 nm (EtOH) [Figure 17]

ESI-MS: M = C20H27BF2N2O2, m/z calculated for [M + H]+: 377.2206, found: 377.2214

[Appendix 29]

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3.2.2 Synthesis of hydrolysed BODIPY

The hydrolysis of the BODIPY framework was conducted with a 0.1 M NaOH-solution, according

to an adjusted literature procedure 42.

500 mg of the previously synthesized BODIPY-framework (1 eq., 1.33 mmol) were dissolved in

15 mL DCM. A mixture of 30 mL methanol and 13 mL of a 0.1 M aqueous NaOH-solution (1 eq.,

1.33 mmol) were added to the solution and stirred for 4 hours at room temperature in the dark.

Afterwards the solvent was evaporated partially. Then 50 mL of ethyl acetate were added and the

mixture was washed with brine (3 x 30 mL) and 1 M HCl (2 x 30 mL). The aqueous phase was re-

extracted again with ethyl acetate (2 x 50 mL). The organic phases were combined and dried over

magnesium sulphate. The solvent was then evaporated with celite to dryness. The product was

purified by DCVC with the same gradient as in the purification of the BODIPY-framework. The

product was obtained as bright red solid with a yield up to 80 % (355.2 mg).

1H-NMR (300 MHz, CDCl3): δ = 1.03-1.08 ppm (t, 3 H, -CH2-CH3), 2.36-2.44 ppm

(q, 4 H, -CH2-CH3), 2.42 ppm (s, 6 H, -CH3), 2.50 ppm (s, 6 H, -CH3), 4.91 ppm (s, 2 H, CH2-OH)

[Appendix 2]

λmax = 538 nm (EtOH) [Appendix 25]

ESI-MS: M = C18H25BF2N2O, m/z calculated for [M + H]+: 335.2101, found: 335.2099

[Appendix 31]

3.2.3 Synthesis of BODIPY-azide via bromination and azidation

The BODIPY-azide was synthesized via a bromination with NBS on one of the 3,5-positions and

a subsequent azidation with NaN3 according to a slightly adjusted literature procedure 55.

100 mg of the previously synthesized hydrolysed BODIPY (1 eq., 0.30 mmol) were dissolved in

3 mL dry DCM. 53 mg of N-bromosuccinimide (1 eq., 0.30 mmol), dissolved in 1 mL DMF were

added and stirred for 1 hour at room temperature in the dark. After this time 150 mg of sodium

azide (7.7 eq., 2.3 mmol), suspended in 3 mL DMF, were added and stirred again for 2 hours in

the dark. The whole procedure was carried out under an inert atmosphere in a glovebox. After the

reaction, 30 mL of ethyl acetate were added to the solution and the mixture was washed with brine

(5 x 25 mL). The organic phase was dried over magnesium sulphate and the solvent was

evaporated with celite to dryness. The product was purified via DCVC with 30 mL fractions and a

gradient with increasing ethyl acetate content in n-heptane (5 x 30:0, 5 x 29:1, 10 x 28:2, 10 x 27:3,

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5 x 26:4, 5 x 25:5, 5 x 24:6, 1 x 20:10). The product was obtained as red precipitate with a yield

up to 29 % (33 mg).


(t, 3 H, -CH2-CH3), 2.42-2.50 ppm (q, 4 H, -CH2-CH3), 2.47 ppm (s, 6 H, -CH3), 2.56 ppm (s, 3 H,

Ar-CH3), 4.55 ppm (s, 2 H, -CH3-N3), 4.97 ppm (s, 2 H, CH2-OH) [Appendix 3]

13C-NMR according to HSQC measurement (75 MHz, CDCl3): δ = 11.99 ppm, 12.49 ppm,

14.02 ppm, 14.53 ppm, 16.82 ppm, 44.76 ppm, 55.94 ppm [Appendix 4]

λmax = 535 nm (MeOH) [Appendix 26]

ESI-MS: M = C18H24BF2N5O, m/z calculated for [M + H]+: 376.2115, found: 376.2111; m/z

calculated for [M - N3]+: 333.1944, found: 333.1956 [Appendix 32]

3.3 Poly(organo)phosphazene synthesis

Three poly(organo)phosphazenes with different substituents were synthesized via an established

procedure 96–98. The starting material for all of these polymers is poly(dichloro)phosphazene, that

is consequently reacted with different sidegroups. The synthesis of the 3-ethynylaniline-BODIPY

substituents is further described in chapter 3.6.5. All reactions were carried out in the glovebox

due to the high reactivity of the poly(dichloro)phosphazene.

3.3.1 Synthesis of the precursor poly(dichloro)phosphazene

The precursor poly(dichloro)phosphazene is synthesized for a polymer chain length of n = 50.

43.3 mg of dichlorotriphenylphosphorane (1 eq., 0.13 mmol) were dissolved in 0.5 mL of dry DCM

and added to a solution of 1.459 g of the monomer (trimethylsilyl)phosphorimidoyl trichloride

(50 eq., 6.5 mmol) in 0.5 mL DCM. The reaction mixture was stirred for 24 hours at room

temperature. The product was directly used for the next step without further purification.

3.3.2 Polymer 1: Mixed macrosubstitution

A mixed macrosubstitution was carried out for the synthesis of Polymer 1 with Jeffamine M-1000

and propargylamine (1:1) as substituents.

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About 30 mL dry THF were added to the precursor solution (1 eq., 6.5 of mmol repeating units).

Then 6.5 g of Jeffamine M-1000 (1 eq., 6.5 mmol) and 1.5 mL of triethylamine (1,6 eq.,

10.7 mmol) in 20 mL of dry THF were added dropwise and stirred for 24 hours at room

temperature. After this time 0.5 mL of propargylamine (1.2 eq., 7.8 mmol) and 1.5 mL of

triethylamine (1.6 eq., 10.7 mmol) in 2 mL dry THF were added and stirred again for 24 hours.

Afterwards the formed salt was filtered off and the solvent was evaporated under reduced

pressure. For purification, dialysis (6-8 kDa) was carried out in ethanol for 72 hours and in Milli-Q-

water for 30 hours. The solvent was then removed on a freeze dryer. The product was obtained

as pale yellow waxy solid with a yield of 38.6 % (2.7567 g).

1H-NMR (300 MHz, CDCl3): δ =1.15 ppm (s, 9 H, -CH-CH3), 2.25 ppm (s, 1 H, -CH2-C-CH),

3.38 ppm (s, 3 H, -O-CH3), 3.38 ppm (s, 2 H, -NH-CH2-C-CH), 3.65 ppm (85 H, -CH-CH2-

/ -CH2-CH2-) [Appendix 5]

31P-NMR (202 MHz, CDCl3): δ = 0.29 ppm (s, -P=N-) [Appendix 7]

Mw = 46035 g mol-1, Đ = 1.273

3.3.3 Polymer 2: Macrosubstitution

For Polymer 2 a macrosubstitution with only propargylamine as the substituent was performed.

About 40 mL dry THF were added to the precursor solution (1 eq., 6.5 mmol of repeating units).

1.08 mL of propargylamine (2.6 eq., 16.9 mmol) and 2.36 mL of triethylamine (2.6 eq., 16.9 mmol)

were dissolved in 5 mL dry THF and added dropwise to the poly(dichloro)phosphazene solution.

The mixture was stirred for 24 hours at room temperature. After this time the solution was filtered

and the solvent was evaporated. Dialysis (1 kDa cut-off) was carried out for 3 hours in Milli-Q-

water, 24 hours in ethanol and 20 hours in chloroform. 405.3 mg (41 %) of the product were

obtained as yellow solid.

1H-NMR (300 MHz, CDCl3): δ = 2.26 ppm (s, 100 H, -CH2-C-CH), 3.58 ppm (s, 100 H, -NH-),

3.78 ppm (s, 200 H, -NH-CH2-C-CH), 7.29-7.76 ppm (m, 15 H, -PPh3) [Appendix 6]

31P-NMR (122 MHz, CDCl3): δ = 0.39 ppm (s, -P=N-), 12.79 ppm (s, Ph3P-) [Appendix 8]

Mw = 12734 g mol-1, Đ = 1.240

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3.3.4 Polymer 3: BODIPY-containing macrosubstitution

For the synthesis of Polymer 3, a mixed macrosubstitution was carried out, where 3-ethynylaniline-

BODIPY and Jeffamine M-1000 in a ratio of 1:9 was used. The synthesis of 3-ethynylaniline-

BODIPY is further described in chapter 3.6.5.

1 mL of a poly(dichloro)phosphazene solution that was synthesized as previously described in

chapter 3.3.1, with a used amount of 0.6 mg of dichlorotriphenylphosphorane (0.0017 mmol) and

20 mg of the monomer (trimethylsilyl)phosphorimidoyl trichloride (0.085 mmol), was used as

starting material.

The precursor solution (1 eq., 0.085 mmol of repeating units) was added to a mixture of 8.4 mg of

3-ethynylaniline-BODIPY (0.2 eq., 0.017 mmol) in 20 mL THF and a few drops triethylamine. The

solution was stirred for 24 hours at room temperature in the dark. After this time 170 mg of

Jeffamine M-1000 (2 eq., 0.17 mmol), dissolved in 3 mL THF, were added, as well as a few drops

triethylamine, and the mixture was stirred for another 24 hours. The resulting solution was filtered,

the solvent was evaporated under reduced pressure and dialysis (6-8 kDa cut-off) was carried out

in ethanol for 4 days and in Milli-Q-water for 5 days. The sample was then freeze dried, yielding

36.8 mg (22 %) of a dark red solid.

Conversion = 73 %

31P-NMR (122 MHz, CDCl3): δ = -10.5 – 14.5 ppm (m, -P=N-, Ph3P-) [Figure 28]

λmax = 533 nm (EtOH)

3.4 Synthesis of poly(ethylene glycol) monomethyl ether azide

The synthesis of poly(ethylene glycol)-azide was performed via a tosylation of the alcohol group,

and a subsequent azidation according to a literature procedure 99.

3.4.1 Tosylation of poly(ethylene glycol) monomethyl ether

3.8 g of p-toluenesulfonyl chloride (10 eq. 20 mmol), were dissolved in 15 mL pyridine. Then, 1 mL

of poly(ethylene glycol) monomethyl ether (1 eq., 2 mmol) were added and stirred for 19 hours at

room temperature under argon-atmosphere. After this time, the reaction mixture was poured into

30 mL of cold water and then extracted with 50 mL DCM. The organic phase was washed with

cold 6 M HCl (2 x 20 mL) and with cold water (3 x 20 mL). The aqueous phases were combined

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and re-extracted with 30 mL DCM. The organic phases were combined and dried over MgSO4.

The solvent was evaporated and 808.0 mg (57 %) of the product were obtained as a clear liquid.

1H-NMR (300 MHz, CDCl3): δ = 2.21 ppm (s, 3 H, -Ar-CH3), 3.12 ppm (s, 3 H, -O-CH3), 3.32 ppm

(s, 6 H, -CH2-CH2-O-CH2-CH2-OTs), 3.40 ppm(s, 40 H, [-O-CH2-CH2-]n), 3.89-3.92 ppm

(t, 2 H, -CH2-OTs), 7.11-7.14 ppm (d, 2 H, Ar-H), 7.53-7.56 ppm (d, 2 H, Ar-H) [Appendix 9]

3.4.2 Azidation of PEG

808.0 mg of the tosylated PEG (1 eq., 1.14 mmol) were dissolved in 10 mL dry DMF. Then 932 mg

of sodium azide (12.5 eq., 14.34 mmol) were added and the reaction mixture was stirred for

40 hours at room temperature. After this time, 25 mL of DCM were added. The mixture was

washed with cold water (5 x 25 mL) and the aqueous phases were combined and reextracted with

25 mL of DCM. The organic phases were combined and dried over MgSO4. The solvent was

evaporated and the product was obtained as slightly yellow liquid with a yield of 83 % (544.4 mg).

1H-NMR (300 MHz, CDCl3): δ = 3.24 ppm (s, 3 H, O-CH3), 3.24-3.27 ppm (t, 2 H, -CH2-N3),

3.51 ppm (s, 46 H, [-O-CH2-CH2-]n) [Appendix 10]

3.5 Amine protection of propargylamine

3.5.1 BOC-protection of propargylamine

The BOC-protection of propargylamine was done, using di-tert-butyl dicarbonate, as can be seen

in Figure 13, according to an adjusted literature procedure 100.

Figure 13: Reaction scheme of the BOC-protection of propargylamine, adapted from literature 100.

2.5 mL of propargylamine (1 eq., 39 mmol) was dissolved in 60 mL dry DCM. The solution was

cooled with ice to 0 °C. Then a solution of 8.5 g of di-tert-butyl dicarbonate (1 eq., 39 mmol) in

20 mL dry DCM were added dropwise via a dropping funnel under argon atmosphere. After

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complete addition of the solution, the ice-bath was removed and the reaction solution was stirred

for 1 h at room temperature. The solution was then extracted with brine (2 x 50 mL) and dried over

MgSO4. The solvent was evaporated, yielding 5.1615 g of a pale yellow crystalline solid (85 %).

1H-NMR (300 MHz, CDCl3): δ = 1.45 ppm (s, 9 H, t-Bu), 2.21-2.23 ppm (t, 1 H, C-H), 3.91 ppm

(broad d, 2 H, CH2), 4.75 ppm (broad s, 1 H, -NH-) [Appendix 11]

3.5.2 Fmoc-protection of propargylamine

The Fmoc-protection was done using Fmoc-OSu, according to an adjusted literature procedure 101

as depicted in Figure 14.

Figure 14: Reaction scheme of the Fmoc-protection of propargylamine, adapted from literature 101.

1 mL of propargylamine (1 eq., 15.6 mmol) was dissolved in 20 mL dry DCM and cooled to 0 °C.

A mixture of 5.26 g Fmoc-Osu (1 eq., 15.6 mmol), 190 mg DMAP (0.1 eq., 1.56 mmol) and 2.7 mL

DIPEA (1 eq., 15.6 mmol) in 30 mL dry DCM were added dropwise via a dropping funnel.

Afterwards, the ice bath was removed and the reaction mixture was allowed to warm up to room

temperature and stirred for 4 hours under argon atmosphere. After this time, the mixture was

extracted with 1 M HCl (1 x 40 mL), sat. NaHCO3 (1 x 40 mL) and brine (1 x 40 mL). The aqueous

phase was re-extracted with 40 mL DCM. The organic phases were combined, dried over MgSO4

and the solvent was evaporated under vacuo, yielding 3.9076 g (90 %) of a white solid.

1H-NMR (300 MHz, CDCl3): δ = 2.26-2.28 ppm (t, 1 H, -CH), 4.00-4.03 ppm (m, 2 H, -NH-CH2-),

4.24 ppm (t, 1 H, -CH-), 4.43-4.46 ppm (d, 2 H, -CH2-O-), 4.98 ppm (s, 1 H, -NH-), 7.30-7.35 ppm

(td, 2 H, Ar-H), 7.39-7.44 ppm (t, 2 H, Ar-H), 7.59-7.62 ppm (d, 2 H, Ar-H), 7.77-7.79 ppm

(d, 2 H, Ar-H) [Appendix 12]

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3.6 Copper-catalyzed azide–alkyne cycloaddition (CuAAC): small

molecules

3.6.1 General procedure

Starting with the BODIPY-azide, four different alkynes were used for this reaction type, following

a general reaction procedure.

1 eq. of the BODIPY-azide was dissolved in 2-3 mL DCM. A mixture of 1.5 eq. of copper sulfate

pentyhydrate and 3 eq. of (+)-sodium-L-ascorbate were dissolved in 2-3 mL Milli-Q-water and

added to the BODIPY-solution. Then about 5-20 eq. of the alkyne was dissolved, if it was a solid,

or directly added. The mixture was stirred overnight under argon atmosphere in the dark. After this

time, 30 mL of DCM were added and the solution was washed with brine (3 x 25 mL) and re-

extracted with 30 mL DCM. The organic phases were combined, dried over MgSO4 and the solvent

was evaporated with celite to dryness. The product was purified using DCVC with increasing ethyl

acetate content in n-heptane.

3.6.2 Synthesis of pentyne-BODIPY

40.5 mg of BODIPY-azide (1 eq., 0.10 mmol) and 213 µL pentyne (20 eq., 2.15 mmol) were used

for the synthesis of pentyne-BODIPY following the general procedure. 46.4 mg (97 %) of the

product were obtained as a red solid.

1H-NMR (300 MHz, CDCl3): δ = 0.74-0.79 ppm (t, 3 H, -CH3), 0.89-0.94 ppm (t, 3 H, -CH3),

1.07-1.12 ppm (t, 3 H, -CH3), 1.60-1.68 ppm (m, 2 H, -CH2-CH3), 2.42 ppm (s, 3 H, -CH3),

2.44-2.48 ppm (m, 4 H, -CH2-CH3), 2.49 ppm (s, 3 H, -CH3), 2.58 ppm (s, 3 H, -CH3),

2.60-2.65 ppm (t, 2 H, -CH2-CH2-CH3), 4.98 ppm (s, 2 H, CH2-OH), 5.76 ppm (s, 2 H, CH2-N-),

7.53 ppm (s, 1 H, -CH-) [Appendix 13]


14.04 ppm, 16.82 ppm, 22.66 ppm, 27.75 ppm, 30.08 ppm, 44.51 ppm, 56.20 ppm, 121.49 ppm

[Appendix 14]

λmax = 533 nm (EtOH)

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3.6.3 Synthesis of BOC-propargylamine BODIPY

34.7 mg of BODIPY-azide (1 eq., 0.09 mmol) and 71.7 mg BOC-propargylamine (5 eq.,

0.46 mmol) were used for the synthesis of BOC-propargylamine-BODIPY following the general

procedure. 33 mg (67 %) of the product were obtained as a viscous red liquid.

1H-NMR (300 MHz, CDCl3): δ = 0.77-0.82 ppm (t, 3 H, -CH3), 1.05-1.10 ppm (t, 3 H, -CH3),

1.39 ppm (s, 9 H, -tBu), 2.36 ppm (s, 3 H, -CH3), 2.40 ppm (m, 4 H, -CH2-CH3), 2.44 ppm (s, 3 H,

-CH3), 2.55 ppm (s, 3 H, -CH3), 4.30-4.32 ppm (d, 2 H, CH2-NH-), 4.89 ppm (s, 2 H, -CH2-OH),

5.13 ppm (s, 1 H, -NH-), 5.74 ppm (s, 2 H, -CH2-N-), 7.77 ppm (s, 1 H, -CH=C-) [Appendix 15]

3.6.4 Synthesis of Fmoc-propargylamine BODIPY

21.1 mg of BODIPY-azide (1 eq., 0.056 mmol) and 78.0 mg Fmoc-propargylamine (5 eq.,

0.28 mmol) were used for the synthesis of Fmoc-propargylamine-BODIPY following the general

procedure. 22.2 mg (60.5 %) of the product were obtained as a bright red solid.


(t, 3 H, -CH2-CH3), 2.40 ppm (s, 6 H, -CH3), 2.34-2.46 ppm (m, 4 H, -CH2-CH3), 2.51 ppm

(s, 3 H, -CH3), 4.09-4.16 ppm (m, 1 H, -CH-), 4.30-4.33 ppm (d, 2 H, -CH2-NH-), 4.40-4.42 ppm

(d, 2 H, -COO-CH2-), 4.85 ppm (s, 2 H, -CH2-OH), 5.43 ppm (s, 1 H, -NH-), 5.75 ppm

(s, 2 H, -CH2-N-), 7.25 ppm (td, 2 H, Ar), 7.36 ppm (t, 2 H, Ar), 7.52 ppm (d, 2 H, Ar), 7.73 ppm (d,

2 H, Ar), 7.80 ppm (s, 1 H, -CH=C-) [Appendix 16]

3.6.5 Synthesis of 3-ethynylaniline-BODIPY

The synthesis of 3-ethynylaniline-BODIPY was done according to a slightly adjusted literature

procedure 55.

20 mg of BODIPY-azide (1 eq., 0.053 mmol) and 1.0 mg copper(I) iodide (0.1 eq., 0.005 mmol)

were dissolved in 7 mL dry DMF. Then 57 µL 3-ethynylaniline (10 eq., 0.53 mmol) were added

and the mixture was stirred for 48 hours at room temperature in the dark. The solution was heated

up to 80 °C and stirred for an additional hour, subsequently, and the progress of the reaction was

checked by TLC. Afterwards, the solution was allowed to cool down to room temperature. Then,

50 mL of ethyl acetate were added, the solution was washed with brine (3 x 40 mL) and the

aqueous phases were re-extracted with 30 mL of ethyl acetate. The organic phases were

Pauline Stadler

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combined, dried over magnesium sulfate and the solvent was evaporated with celite to dryness.

The product was purified with DCVC with increasing ethyl acetate content in n-heptane. 8.4 mg

(32 %) of the product was obtained as red solid.


(t, 3 H, -CH2-CH3), 2.42 ppm (s, 3 H, -CH3), 2.42-2.49 ppm (m, 4 H, -CH2-CH3), 2.49 ppm (s, 3 H,

-CH3), 2.60 ppm (s, 3 H, -CH3), 3.68 ppm (broad s, 2 H, -NH2), 4.99 ppm (s, 2 H, -CH2-OH),

5.83 ppm (s, 2 H, -CH2-N-), 6.67-7.15 ppm (m, 4 H, Ar-H), 8.00 ppm (s, 1 H, -CH=C-)

[Appendix 17]

3.7 Copper-catalyzed azide–alkyne cycloaddition (CuAAC):

macromolecules

3.7.1 General procedure

During the CuAAC with the poly(organo)phosphazenes, the BODIPY-azide was reacted with the

terminal triple bond of the propargylamine substituent, forming a triazole ring. Polymer 1 and

Polymer 2 were used for this reaction, their chemical structure and synthesis is described in

chapter 3.3.2 and 3.3.3.

1 eq. of the poly(organo)phosphazene and between zero and two eq. of the azide, depending on

the wanted substitution of triplebonds, were dissolved in DCM. 1.5 eq. of copper sulfate

pentahydrate and 3 eq. of (+)-sodium-L-ascorbate were dissolved in the same amount of Milli-Q-

water to get a 1:1 mixture of DCM and H2O. The components were mixed and stirred at room

temperature in the dark. After a certain reaction time, 30 mL of DCM were added and the solution

was extracted with brine (3 x 25 mL). The aqueous phases were re-extracted with 30 mL DCM.

The organic phases were combined, dried over magnesium sulfate and the solvent was

evaporated under vacuo. Purification was performed via ultrafiltration with Vivaspin-20

ultrafiltration tubes (MWCO = 3 kDa, 5000 g) with an ethanol / water (1:1) solution and

subsequent dialysis (6-8 kDa cut-off) in ethanol. The purity of the product was verified via aqueous

GPC-measurement, with the UV-vis detector adjusted to a wavelength of 540 nm to detect the

BODIPY-compound. The concentration of the reacted BODIPY on the polymer was measured via

a UV-vis calibration of the pentyne-BODIPY.

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3.7.2 2.7 w% BODIPY-azide on Polymer 1

17.7 mg BODIPY-azide (0.5 eq., 0.047 mmol) and 103.6 mg of the Polymer 1 (1 eq., 0.094 mmol

of repeating units) were used for this synthesis according to the general procedure. The reaction

took place over the course of 24 hours. A red solid was obtained as product after purification. The

concentration of the reacted BODIPY on the polymer according to the UV-vis calibration was

2.7 w% of BODIPY. Conversion = 16 %.


3.7.3 9.6 w% BODIPY-azide on Polymer 1

33.4 mg BODIPY-azide (1 eq., 0.089 mmol) were used for the reaction with 97.7 mg of Polymer 1

(1 eq., 0.089 mmol of repeating units), according to the general procedure. The reaction was

carried out over the course of 3 days. After purification, the product was obtained as dark red solid.

Through the UV-vis measurement, a BODIPY content of 9.6 w% was calculated.

Conversion = 31 %.


3.7.4 PEG-azide on Polymer 2

100 mg of PEG-azide (0.5 eq., 0.174 mmol) were used for the reaction with 53 mg Polymer 2

(1 eq., 0.348 mmol of repeating units) to substitute 25 % of the triple bonds. The reaction was

done according to the general procedure over the course of 24 hours, but with a lower amount of

catalyst, where 4.3 mg of copper sulfate pentahydrate (0.05 eq., 0.017 mmol) and 6.9 mg of

(+)-sodium-L-ascorbate (0.1 eq., 0.035 mmol) were used. Extraction was done as described, but

no further purification was carried out. The product was obtained as yellow viscous liquid and

directly used for further reaction, assuming complete reaction.

3.7.5 BODIPY-azide on 25 % PEG-substituted Polymer 2

90 mg of the PEG-substituted polyphosphazene (1 eq., 0.204 mmol of repeating units) from

chapter 3.7.4 were used for the CuAAC with 23 mg of the BODIPY-azide (0.3 eq., 0.061 mmol)

according to the general procedure with a reaction time of 3 days. As in chapter 3.7.4, lower

amounts of catalyst with 2.5 mg copper sulfate pentahydrate (0.05 eq., 0.01 mmol) and 4.0 mg

(+)-sodium-L-ascorbate (0.1 eq., 0.02 mmol) were used. The product was extracted as described

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above and purification was altered to a single dialysis (3.5 kDa cut-off) in ethanol for 24 hours.

The solvent was then evaporated, yielding 75.9 mg of the product as dark red solid. Conversion =

54 %.

λmax = 535 nm (EtOH) [Appendix 28]

3.8 Coupling methods testing

Two different procedures were conducted according to literature, reaction with an acid chloride 102

and N,N′-dicyclohexylcarbodiimide (DCC) catalyzed coupling with an acid 103, respectively.

3.8.1 Reaction of the hydrolysed BODIPY with phenoxyacetyl chloride

The reaction was carried out under an inert atmosphere. 85.7 mg of the hydrolysed BODIPY

(1 eq., 0.25 mmol) were dissolved in 3 mL dry DCM. 232 mg of DIPEA (7 eq., 1.79 mmol) in 1 mL

DCM were added and cooled for 5 minutes in the freezer. Then 43.7 mg of phenoxyacetyl chloride

(1 eq., 0.25 mmol) in 1 mL DCM were added dropwise to the solution. The reaction mixture was

stirred for 4 hours at room temperature in the dark. After this time, 30 mL of DCM were added and

the solution was washed with sat. NaHCO3 (2 x 25 mL) and brine (1 x 25 mL). The aqueous

phases were combined and re-extracted with 30 mL of DCM. The organic phases were dried over

magnesium sulfate and the solvent was evaporated with celite to dryness. The product was

purified via DCVC with 30 mL fractions and increasing ethyl acetate content in n-heptane with a

1 mL increase of ethyl acetate every second fraction. The product was obtained as red solid

(62.9 mg, yield = 52 %).

1H-NMR (300 MHz, CDCl3): δ = 0.99-1.04 ppm (t, 6 H, -CH2-CH3), 2.19 ppm (s, 6 H, -CH3),

2.32-2.39 ppm (q, 4 H, -CH2-CH3), 2.48 ppm (s, 6 H, -CH3 ), 4.67 ppm (s, 2 H, CH2-O-), 5.43 ppm

(s, 2 H, CO-CH2-), 6.85-6.88 ppm (td, 2 H, Ar-H), 6.94-6.99 ppm (tt, 1 H, Ar-H), 7.22-7.28 ppm

(td, 2 H, Ar-H) [Appendix 18]

ESI-MS: M = C26H31BF2N2O3, m/z calculated for [M + H]+: 469.2469, found: 469.2487

[Appendix 33]

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3.8.2 Reaction of the hydrolysed BODIPY with benzoic acid

50.0 mg of hydrolysed BODIPY (1 eq., 0.150 mmol) were dissolved in 40 mL dry DCM and cooled

to 0 °C. 18.2 mg of benzoic acid (1 eq., 0.150 mmol) in 1 mL DCM were added. Afterwards

37.0 mg DCC (1.2 eq., 0.179 mmol) and 1.8 mg of DMAP (0.1 eq., 0.015 mmol) in 1 mL DCM

were added and the mixture was stirred for 15 minutes. Then, the ice bath was removed, the

reaction mixture was allowed to warm up to room temperature and stirred for 2 days at room

temperature in the dark. The progress of the reaction was checked with TLC, therefore, after this

time, another 25.0 mg of DCC (0.8 eq., 0.096 mmol) were added and stirred again for 5 days. The

solvent was then evaporated with celite to dryness and the product was purified via DCVC with

30 mL fractions with increasing ethyl acetate content in n-heptane with a 1 mL increase of ethyl

acetate every second fraction. 12.9 mg (20 %) of the product were obtained as a red solid.

1H-NMR (300 MHz, CDCl3): δ = 1.03-1.08 ppm (t, 6 H, -CH2-CH3), 2.28 ppm (s, 6 H, -CH3),

2.36-2.44 ppm (q, 4 H, -CH2-CH3), 2.54 ppm (s, 6 H, -CH3 ), 5.55 ppm (s, 2 H, CH2-O-),

7.43-7.48 ppm (td, 2 H, Ar-H), 7.57-7.62 ppm (tt, 1 H, Ar-H), 8.06-8.08 ppm (d, 2 H, Ar-H)

[Appendix 19]

3.9 Model compound: reaction of the photoreactive OH-group

Two different coupling methods were used to obtain a model compound to test the photorelease

on the meso-position. The same methods as for the hydrolysed BODIPY were adapted for the

usage of pentyne-BODIPY.

3.9.1 Reaction of pentyne-BODIPY with phenoxyacetyl chloride

The reaction was done under an inert atmosphere in the glovebox. 46.4 mg of pentyne-BODIPY

(1 eq., 0.105 mmol) were dissolved in 5 mL dry DCM. 94.7 mg DIPEA (7 eq., 0.733 mmol) in 1 mL

DCM were added and the mixture was cooled in the freezer for 5 minutes. Then 17.9 mg of

phenoxyacetyl chloride (1 eq., 0.105 mmol) in 1 mL DCM were added dropwise and the mixture

was stirred for 4 hours at room temperature. After this time, 30 mL of DCM were added and the

solution was washed with sat. NaHCO3 (2 x 25 mL) and brine (1 x 25 mL). The aqueous phases

were combined and re-extracted with 30 mL of DCM. The organic phases were dried over MgSO4

and the solvent was evaporated with celite under reduced pressure. The product was purified via

DCVC with 30 mL fractions with increasing ethyl acetate content in n-heptane with a 1 mL increase

of ethyl acetate every second fraction. 2.6 mg (4.3 %) of the product were obtained as a red solid.

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(t, 3 H, -CH2-CH2-CH3), 1.06-1.10 ppm (t, 3 H, -CH3), 1.60-1.68 ppm (m, 2 H, -CH2-CH2-CH3),

2.33 ppm (s, 3 H, -CH3), 2.39 ppm (s, 3 H, -CH3), 2.44-2.46 ppm (m, 4 H, -CH2-CH3), 2.57 ppm

(s, 3 H, -CH3), 2.60-2.65 ppm (t, 2 H, -CH2-CH2-CH3), 4.76 ppm (s, 2 H, -CH2-O-), 5.39 ppm

(s, 2 H, -CO-CH2-) 5.77 ppm (s, 2 H, CH2-N-), 6.93-6.95 ppm (m, 2 H, Ar-H), 6.00-7.07 ppm

(m, 2 H, Ar-H), 7.31-7.34 ppm (m, 2 H, Ar-H), 7.54 ppm (s, 1 H, -CH-) [Appendix 20]

3.9.2 Reaction of pentyne-BODIPY with phenylacetic acid

41.2 mg of pentyne-BODIPY (1 eq., 0.093 mmol) were dissolved in 20 mL dry DCM. 38.3 mg of

DCC (2 eq., 0.185 mmol), 1.1 mg of DMAP (0.1 eq., 0.009 mmol) and 18.9 mg of phenylacetic

acid (1.5 eq., 0.139 mmol), dissolved in 3 mL dry DCM, were added to the BODIPY-solution. The

reaction mixture was stirred at room temperature under argon atmosphere in the dark. The

progress of the reaction was checked with TLC. After 22 hours of stirring, the reaction was

completed and the solvent was evaporated with celite under vacuo. The product was purified via

DCVC with increasing ethyl acetate content in n-heptane, obtaining a red solid (24.6 mg,

47 % yield)


(t, 3 H, -CH2-CH3), 1.04-1.09 ppm (t, 3 H, -CH3), 1.59-1.72 ppm (m, 2 H, -CH2-CH3), 2.09 ppm

(s, 3 H, -CH3), 2.16 ppm (s, 3 H, -CH3), 2.38-2.47 ppm (m, 4 H, -CH2-CH3), 2.56 ppm

(s, 3 H, -CH3), 2.64-2.66 ppm (t, 2 H, -CH2-CH2-CH3), 3.70 ppm (s, 2 H, CO-CH2-), 5.34 ppm

(s, 2 H, -CH2-O-), 5.76 ppm (s, 2 H, CH2-N-), 7.28-7.35 ppm (m, 5 H, Ar-H), 7.54 ppm

(s, 1 H, -CH-) [Appendix 21]


16.82 ppm, 22.15 ppm, 27.75 ppm, 41.21 ppm, 44.77 ppm, 57.98 ppm, 120.99 ppm, 127.34 ppm,

128.86 ppm [Appendix 22]

ESI-MS: M = C31H38BF2N5O2, m/z calculated for [M - pentyne]+: 451.2363, found: 451.2389

[Appendix 34]

λmax = 540 nm (EtOH) [Appendix 27], (λ540) = 42100

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3.10 Reaction of BODIPY-azide with phenylacetic acid

27.7 mg of BODIPY-azide (1 eq., 0.074 mg) were dissolved in 20 mL dry DCM. Then, 30.4 mg of

DCC (2 eq., 0.147 mmol), 1.0 mg of DMAP (0.1 eq., 0,007 mmol) and 15.0 mg of phenylacetic

acid (1.5 eq., 0.139 mmol), dissolved in 3 mL dry DCM, were added and the mixture was stirred

at room temperature under argon atmosphere. The progress of the reaction was checked with

TLC, where after 18 hours a high consumption of the educt could be seen. The solvent was

evaporated with celite under reduced pressure and the product was purified via DCVC with ethyl

acetate and n-heptane as eluents. 25.1 mg (69 %) of the product were obtained as red solid.


(t, 3 H, -CH2-CH3), 2.13 ppm (s, 6 H, -CH3), 2.35-2.41 ppm (q, 2 H, -CH2-CH3), 2.43-2.49 ppm (q, 2

H, -CH2-CH3), 2.57 ppm (s, 3 H, -CH3), 3.71 ppm (s, 2 H, CO-CH2-), 4.56 ppm (s, 2 H, CH2-N3-),

5.33 ppm (s, 2 H, -CH2-O-), 7.28-7.32 ppm (m, 5 H, -Ar) [Appendix 23]


15.04 ppm, 16.59 ppm, 40.92 ppm, 45.12 ppm, 58.17 ppm, 127.17 ppm, 128.72 ppm

[Appendix 24]

ESI-MS: M = C31H38BF2N5O2, m/z calculated for [M – N3]+: 451.2363, found: 451.2381

[Appendix 35]

3.11 Loading on polymer

3.11.1 CuAAC of phenylacetic acid-BODIPY-azide with Polymer 1

21.8 mg BODIPY-azide with phenylacetic acid (0.75 eq., 0.044 mmol) were used for the reaction

with 64.7 mg poly(organo)phosphazene with Jeffamine M-1000 and propargylamine (1:1)

substituents (Polymer 1). The reaction and purification were done according to the general

procedure in chapter 3.7.1, where the reaction was carried out over the course of 3 days.

Conversion = 33 %.

3.11.2 Loading of phenylacetic acid on Polymer 1 bearing 9.6 w% BODIPY

53.9 mg of the previously synthesized BODIPY-containing polymer, chapter 3.7.3, were used for

the loading with phenylacetic acid. The polymer, containing about 5.1 mg BODIPY (1 eq.,

0.014 mmol), was dissolved in 20 mL dry DCM. Then 12.7 mg DCC (4.5 eq., 0.062 mmol),

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1 mg DMAP (cat.) and 7.0 mg phenylacetic acid (4 eq., 0.052 mmol) were added and the reaction

was stirred for 7 days at room temperature under argon atmosphere. After this time, the solvent

was evaporated under reduced pressure and dialysis (6-8 kDa cut-off) was performed in ethanol

for 48 hours and in water for 8 hours. The sample was then freeze dried, yielding a dark red solid.

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4 Results and discussion

4.1 BODIPY synthesis and modification

4.1.1 Synthesis of the BODIPY framework

The BODIPY framework is synthesized from kryptopyrrole and acetoxyacetyl chloride, followed by

a complexation with boron trifluoride diethyl etherate according to the reaction scheme in

Figure 15.

Figure 15: Reaction scheme of the BODIPY-framework synthesis, adapted from literature 42.

The main obstacles of the synthesis of the BODIPY framework was the low yield and its

purification, still showing impurities after column chromatography, despite its good separation in

the column. To improve the synthesis to obtain higher yields and higher purity, different conditions

were altered, especially the equivalences of the starting materials. Side products of the reaction

were studied in order to get a better understanding of the synthesis.

In the possible reaction mechanism of the BODIPY formation, shown in

Figure 16, it is suggested, that in a first step, one kryptopyrrole reacts with the acetoxyacetyl

chloride, forming 2-(4-ethyl-3,5-dimethyl-1H-pyrrol-2-yl)2-oxoethyl acetate after cleavage of

hydrogen chloride and a proton transfer. This intermediate was detected as a major impurity in the

product fraction of the BODIPY, even after purification. It can be easily distinguished from the

product, due to a peak in the 1H-NMR at 5.06 ppm, whereas the BODIPY framework shows a

dominant peak at 5.33 ppm. The detection of this intermediate is an indication that the reaction

was not fully completed or the equilibrium limits further consumption of the kryptopyrrole. In an

optimization process of this synthesis, the conditions were adjusted from an excess of

acetoxyacetyl chloride of 1.2 eq. in the beginning to an excess of kryptopyrrole of 2.2 eq., leading

to an increase in yield from about 15-20 % to 35-40 %. The reason most probably lies in the

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second reaction step of the proposed BODIPY reaction mechanism. A second kryptopyrrole reacts

with the previously formed intermediate, constituting an aromatic dipyrrine system after cleavage

of water. Therefore, with the usage of an increased amount of kryptopyrrole, the equilibrium can

be shifted to the product side. A higher excess of kryptopyrrole of 2.5 eq. decreases the yield

again to about 30 %. Due to the fact that the product mostly still contains the intermediate of the

reaction, the yields were calculated via the 1H-NMR ratios of the product and the intermediate and

used for follow-up reaction without further purification. In the last step of the BODIPY formation

in situ complexation with boron trifluoride diethyl etherate is carried out in the presence of a base,

specifically triethylamine. During this synthesis step, the high excess of the BF3 is crucial. It was

also tested to use only 4 eq. of BF3 · OEt2, but yields dramatically decreased. Despite the usage

of such a high excess of boron trifluoride, the reaction is extremely sensitive towards moisture. In

literature 104 the effect of water on the boron centrum is described, where the excess of BF3 leads

to an activation of the B-F bond of the produced BODIPY framework allowing a nucleophile, such

as water, to attack the boron atom, followed by the cleavage of the BF2 moiety. Hence, anhydrous

conditions have to be utilized for the synthesis of the BODIPY framework, otherwise loss of the

BF2 group leads to the formation of dipyrrine, as well as boronic acid and BF4 anions. Furthermore,

this method of masking and demasking of BODIPY should be remembered, when nucleophilic or

reductive functionalizations are done on the BODIPY core 105.

Figure 16: Possible mechanism of the BODIPY formation.

The recorded UV-vis spectrum in methanol, shown in Figure 17, is in agreement with previously

recorded data 106. The main absorbance peak has its maximum at 544 nm, which is in the region

of green light in the visible spectrum. This arises from the S0-S1 transition. The ethyl groups on the

C2 and C6 positions act as electron donating groups that stabilize the LUMO level and therefore

decrease the energy gap. In addition, the acetyl group in the meso-position stabilizes the LUMO

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due to a +I effect. In the spectrum also a vibronic shoulder in the range of about 490-520 nm can

be seen and a broad weak absorption near the UV-range, which arises from S0-S2

transitions 106,107,108. The overlapping of the absorbance and the excitation spectra allows

predictions of the purity of the compound. The emission spectrum expectedly behaves like a

mirror-image of the absorbance spectrum and has its maximum at 564 nm.

Figure 17: UV-vis spectrum of the BODIPY-framework in methanol.

The ESI-MS spectrum [Appendix 29] revealed that the highest peak with a m/z ratio of 377.2214

is the [M + H]+ peak, which is typical for a soft ionization technique like ESI. When zoomed in on

the basepeak [Appendix 30], the isotope pattern of the boron can be found, where the peak at

376.2236 with a height of 23.51 % in comparison to the base peak correlates to the 10B isotope,

which has a natural abundance of about 20 %. The peak at 378.2259 with a height of 22.22 %,

compared to the base peak, arises from the 13C isotope. In addition the [M + Na]+ and the [M + K]+

peak are visible, common for this ionization method. However, another high peak at 357.2150

corresponds to the hydrolysed BODIPY, which means that the BODIPY fragmented, even though

a soft ionization method was used. This demonstrates that the acetyl moiety is surprisingly labile

544

564

350 400 450 500 550 600 650 700 750

0.0

0.2

0.4

0.6

0.8

1.0

norm

aliz

ed

In

ten

sity / a

.u.

wavelength / nm

Absorbance

Emission (485 nm)

Excitation (595 nm)

Pauline Stadler

39

and can be easily cleaved off. Another peak could be assigned to the [M - BF2]+, indicating that

the complexation with boron is not too stable.

The X-Ray structure analysis, illustrated in Figure 18, shows that the boron atom is coordinated in

a tetrahedral geometry by the fluorine and nitrogen atoms with angles between 107° and 111°.

The bond lengths between the B-F bonds are with 1.533 Ǻ and 1.534 Ǻ longer than the B-N bonds

with a length of 1.386 and 1.388 Ǻ. This can be the result of the high electronegativity of the

fluorine atoms (R-Factor = 6.6 %). All other crystal data of the X-Ray structure analysis of the

BODIPY-framework in comparison to the BODIPY-azide are listed in Table 2.

Figure 18: X-Ray structure analysis of the BODIPY-framework.

4.1.2 Synthesis of the hydrolysed BODIPY

The acetyl group of the BODIPY framework was hydrolysed using an aqueous 0.1 M NaOH-

solution according to the general reaction scheme in Figure 19.

Figure 19: Reaction scheme of the BODIPY hydrolysis, according to literature 42.

Pauline Stadler

40

Mild conditions are preferred for the hydrolysis of the BODIPY framework due to the low stability

of the compound. Therefore, the usage of 1 eq. of 1 M aq. NaOH-solution results in significantly

higher yields, compared to the utilization of 5 eq. of LiOH. The solvent choice for this synthesis

step with DCM and methanol is crucial to enable miscibility of the organic and the aqueous phase,

necessary to get a high conversion. The DCM is used to enable solubility of the BODIPY

framework, whereas the methanol helps to avoid phase separation.

The conversion of the BODIPY framework to the hydrolysed BODIPY can easily be seen in the

1H-NMR, where the peak at 5.33 ppm corresponding to the methylene group in the meso-positions

shifts to 4.91 ppm, as it can be seen in Figure 21. Furthermore, the acetyl peak at 2.15 ppm

disappears and the signal of the CH3 groups in the 3,5-positions shift from 2.27 ppm to 2.42 ppm

(see Appendix 1 and Appendix 2).

The UV-vis spectrum of the hydrolysed BODIPY [Appendix 25] looks similar to the one of the

BODIPY framework with slightly shifted maxima. In detail the absorption maximum is shifted from

544 nm (EtOH) to 538 nm (MeOH) for the BODIPY-framework and the hydrolysed compound,

respectively (Figure 22). So the -I effect of the hydroxyl group destabilizes the LUMO, leading to

a higher energy gap, resulting in a shorter absorption wavelength 106. Despite this, also the solvent

choice can slightly influence the absorption maximum. The maximum of the emission wavelength,

in accordance with the the absorbance maxima, is hypsochromically shifted from 564 nm to

557 nm.

In the ESI-MS spectrum [Appendix 31], the highest peak with a m/z ratio of 335.2099 corresponds

to the [M + H]+ peak. Also the [M + K]+ peak can be seen, as well as the typical isotope pattern of

the basepeak. Curiously, the peak at 315.2037 could be assigned to the [M - F]+, assuming that a

fluorine atom can be cleaved even under soft ionization conditions.

Pauline Stadler

41

4.1.3 Synthesis of BODIPY-azide

The BODIPY-azide was synthesized via a bromination with NBS on one of the 3,5-positions and

a subsequent azidation with NaN3 according to the reaction scheme in Figure 20.

Figure 20: Reaction scheme of the BODIPY bromination and azidation, adapted from literature 55.

The synthesis of the BODIPY-azide was slightly optimized compared to the literature procedure 55

by the usage of DMF instead of DCM to dissolve the NBS. Hence, the yield could be increased by

~5 %. The influence of the reaction times on the bromination and the azidation was tested as well,

where it was found, that longer reaction times of 2 hours at the bromination step does not influence

the yield. Surprisingly, the extension of the reaction time at the azidation step from 1 to 2 hours

could improve the reaction yield slightly. In general, NBS as bromination source reacts due to a

radical mechanism. In this synthesis the reaction is carried out without any initiator and in absence

of light, where the mechanism is more likely to be an electrophilic substitution 55. Since in the first

step only the mono-brominated compound could be observed, the hydrolysed BODIPY must have

a higher reactivity for the bromination than the mono-brominated compound. Otherwise the

di-brominated compound would lead to a di-azide functionalized BODIPY, which was never

observed. The conversion of this synthesis is relatively low with significant amounts of educt left

Pauline Stadler

42

unreacted. However, not more than 1 eq. of NBS was used, to avoid any di-functionalized products

and crosslinking during the CuAAC reaction. During the purification step a yellow glowing fraction

could be observed quite often, which was characterized via 1H-NMR and MS as Methyl-BODIPY.

Here the alcohol moiety in the meso-position disappeared and the CH2 group was converted to

CH3. The mechanism for this is could not be elucidated, but could be a result of photodegradation.

In Figure 21 the main differences of the chemical shifts of the 1H-NMR of the BODIPY framework,

the hydrolysed BODIPY and the BODIPY-azide can be seen. Especially the conversion of the

symmetrical hydrolysed BODIPY to the asymmetrical BODIPY-azide can be seen through the

splitting of the protons at the respective positions. The two protons beside the azide functionality

shift from 2.50 ppm to 4.55 ppm. Additionally, the doublets and triplets of the ethyl-functionality at

positions 2 and 6 are split into a doublet of doublets and a doublet of triplets, respectively.

Figure 21: Comparison of the 1H-NMR spectra of the BODIPY framework (black), the hydrolysed BODIPY

(blue) and the BODIPY-azide (red).

Pauline Stadler

43

The UV-vis spectrum [Appendix 26] looks very similar to the spectra of the BODIPY-framework

and the hydrolysed BODIPY. Comparisons of the absorbance spectra of the BODIPY compounds

can be seen in Figure 22.The UV-vis absorbance spectrum has its maximum at 535 nm, which is

slightly hypsochromically shifted compared to the hydrolysed BODIPY. As described in

literature 106, asymmetrical compounds show higher band gaps, which could explain this shift.

However, in this literature work, only 2,6-substituted BODIPY structures were considered,

therefore this explanation may not be applicable for the structure described here. The fluorescence

maximum stayed similar to the hydrolysed BODIPY.

Figure 22: Absorbance spectra of the BODIPY framework (black), the hydrolysed BODIPY (blue) and the

BODIPY-azide (red) with zoomed spectra of the maxima.

In the ESI-MS spectrum [Appendix 32] the highest peak with an m/z ratio of 333.1956 can be

assigned to the [M - N3]+ peak, indicating that the azide-functionality can be cleaved rather easily

at this position. Alongside this dominant peak, also some other peaks could be assigned to the

[M + H]+, the [M + Na]+ and the [M - F - N3]+. The typical isotope pattern of the boron and the

carbon were visible for the basepeaks as well.

Pauline Stadler

44

In the FT-IR spectrum of the BODIPY-azide in Figure 23, the azide stretching can be seen as a

strong peak at about 2100 cm-1. In the region of 2900-3000 cm-1 the C-H stretching of alkanes and

alkenes are visible. A strong peak at 3545 cm-1 can be assigned to the O-H stretching of the free

alcohol moiety in the meso-position.

Figure 23: FT-IR spectrum of the BODIPY-azide.

The X-Ray structure analysis, illustrated in Figure 24, shows the structure of the BODIPY-azide.

Surprisingly, the azide functionality is coordinated away from the planar BODIPY core with angles

of 116.3° between the carbon atom and the two nitrogen atoms and 172.1° between the three

nitrogen atoms of the azide moiety. The bond lengths of the B-F bonds are slightly increased to

1.395 Ǻ and 1.409 Ǻ in comparison to the BODIPY-framework. The bond lengths of the B-N bonds

are close to the one of the BODIPY-framework with 1.539 and 1.526 Ǻ. An R-factor of 5.83 % was

achieved for this measurement. Disagreement with calculated models may arise from the flexibility

of the N-N bonds. Other relevant data of the crystal structure of the BODIPY-azide are listed in

Table 2.

4000 3500 3000 2500 2000 1500 1000 500

0.0

0.2

0.4

0.6

0.8

1.0

norm

aliz

ed tra

nsm

itta

nce

wavenumber / cm-1

N=

N=

N s

tretc

hin

g

O-H

stre

tchin

g

(free a

lcoh

ol)

C-H

stre

tchin

g (A

lken

e)

C-H

stre

tchin

g (A

lkan

e) C=

C s

tretc

hin

g

(cyclic

alk

ene)

C=

C b

end

ing

(alk

ene)

Pauline Stadler

45

Figure 24: X-Ray structure analysis of BODIPY-azide.

Table 2: X-ray crystal data, data collection and structure refinement for structures of the BODIPY framework

and -azide.

Compound BODIPY framework BODIPY-azide

Empirical formula C20H27BF2N2O2 C18H24BF2N5O

Formula weight (g mol-1) 376.24 375.23

Crystal system monoclinic triclinic

Space group C2/c P1̅

Temp (K) 293 293

a (Å) 17.1373(13) 8.7126(10)

b (Å) 11.7136(9) 10.0034(12)

c (Å) 20.6062(15) 11.8736(14)

α () 90 69.381(2)

β () 99.0463(10) 80.082(2)

γ () 90 76.862(2)

V (Å3) 4085.03 938.372

Z 8 2

ρcalc (g cm-3) 1.224 1.328

Reflns collected 21027 11484

Indep. reflns 3500 4017

Obs. reflns [I > 2σ(I)] 2485 2534

Param. refin./ restr. 251 / 0 250 / 0

Absorption correction multi-scan multi-scan

R1 0.066 0.058

wR2 0.197 0.190

Pauline Stadler

46

4.2 Poly(organo)phosphazene synthesis

4.2.1 Synthesis of the precursor poly(dichloro)phosphazene

The poly(organo)phosphazenes were synthesized, starting from the precursor

poly(dichloro)phosphazene, whose reaction scheme is shown in Figure 25 97,98.

Figure 25 :Reaction scheme of the poly(dichloro)phosphazene synthesis with n = 50.

4.2.2 Polymer 1: mixed macrosubstitution

For the synthesis of Polymer 1, a mixed macrosubstitution was employed, using Jeffamine M-1000

and propargylamine as substituents, as it can be seen in the reaction scheme in Figure 26. The

Jeffamine is therefore responsible for water-solubility, while the propargylamine is considered as

reactive moiety for further reactions.

Figure 26: Reaction scheme of the macrosubstitution of poly(dichloro)phosphazene with Jeffamine-M-1000

and propargylamine (1:1) with x = 3, y = 19 and n = 50.

Pauline Stadler

47

Full substitution of the chlorine atoms can be deduced from the 31P-NMR, [Appendix 7], with an

intense peak at around 0.3 ppm. In contrast, the poly(dichloro)phosphazene gives a sharp,

characteristic peak at -18 ppm which is absent after the macrosubstitution 96. The phosphorus at

the endgroup, substituted with three phenyl-rings and expected at about 15 ppm 96, cannot be

seen, since the noise is too high. The 1H-NMR [Appendix 5] is in agreement with previously

recorded data 96.

The molecular weight of the poly(organo)phosphazene with Jeffamine M-1000 and

propargylamine substituents (1:1) with a theoretical length of 50 repetition units was determined

via SEC against polystyrene standards to be Mn = 36.2 kDa with a dispersity of 1.2. Since the SEC

does not consider aggregation or branching of the polymers, these results are only a rough

estimation and vary from the theoretical molecular mass. The dispersity of this polymer is quite

low, despite the various molecular lengths of the Jeffamine M-1000 sidegroup.

In the FT-IR spectrum in Appendix 36, the C-H stretching of alkanes at 2880 cm-1 is visible, as

well as a small and broad N-H stretching peak at about 3200 cm-1 of the secondary amine. In the

fingerprint region the P=N stretching at 1240 cm-1 and the intense C-O ether stretching at 1100

cm-1 could be assigned. No triple bond of the propargylamine substituent can be seen in the

spectrum, probably due to the fact that it is relatively small in relation to the Jeffamine.

4.2.3 Polymer 2: macrosubstitution

Polymer 2 is a poly(organo)phosphazene with only propargylamine substituents, as can be seen

in the reaction scheme in Figure 27. It was synthesized to allow for more precise analytics after a

copper-catalyzed azide-alkyne cycloaddition with the BODIPY-azide compared to Polymer 1.

Since in the FT-IR of Polymer 1, the triple bonds cannot be seen, no estimation about the amount

of BODIPY on the polymer can be made this way. Hence, the propargylamine polymer would allow

an easy and fast prediction of the amount of reacted BODIPY. Water-solubility should be then

achieved by a reaction with PEG-azide. However, the CuAAC, according to the general procedure,

described in chapter 3.7.1, did not lead to the desired product, neither with the BODIPY-azide nor

with the PEG-azide. A reason for this could be the high amount of copper, which can lead to

complexations. Due to the fact, that only small amounts of BODIPY-azide could be synthesized,

other synthesis procedures with smaller amounts of catalyst were tested with the PEG-azide,

whose results are described in chapter 4.5.2. Following these initial tests, the reaction was carried

Pauline Stadler

48

out with similar conditions with the BODIPY-azide, described in chapter 4.5.3. Further synthesis

procedures were not tested, due to the limited stability of this polymer and some solubility issues.

Figure 27: Reaction scheme of the macrosubstitution of poly(dichloro)phosphazene with propargylamine

with n = 50.

The 31P-NMR [Appendix 8] of Polymer 2 shows complete substitution of the chlorine atoms with a

peak at 0.37 ppm. The peak of the phosphine endgroup of the polymer is visible at 12.88 ppm.

The 1H-NMR is in agreement with previously recorded data 96. With the ratio of the integrals of the

peaks corresponding to the endgroup and the substitutents, respectively, the length of the polymer

could be estimated to be around 50 repeating units, agreeing with the targeted value.

The GPC measurement of this polymer predicted a Mn of 10.3 kDa, roughly comparable to the

calculated value. As described in chapter 4.2.2, this value is just an estimation based on a

calibration against linear polystyrene standards.

The FT-IR spectrum in Appendix 37 shows a strong band of the alkyne at 3290 cm-1 and a low

one at 2120 cm-1. The P=N stretching of the backbone of the poly(organo)phosphazene is visible

at 1190 cm-1.

4.2.4 Polymer 3: BODIPY-containing macrosubstitution

For the synthesis of Polymer 3, a mixed macrosubstitution with 3-ethynylaniline-BODIPY

(described in chapter 3.6.5 and 4.4.3) and Jeffamine M-1000 in a ratio of 1:9 was carried out.

Polymer 3 was synthesized to try a direct conjugation of the BODIPY onto the polyphosphazene

Pauline Stadler

49

chain, circumventing the need of a CuAAC on the polymer. Furthermore, the substitution of the

chlorine on the poly(dichloro)phosphazene is known to perform fast and to completion, allowing

higher control over the loading of BODIPY on the polymer 80.

In the 1H-NMR only the Jeffamine sidegroup can be seen, due to the fact, that it has a much higher

number of protons than the 3-ethynylaniline-BODIPY. The 31P-NMR spectrum reveals a high

signal to noise ratio even though a high number of scans was set, because only a low product

concentration could be achieved since only a small-scale approach was conducted due to limited

amount of the 3-ethynylaniline-BODIPY. A broad multiplet in the range of -10.5 to 14.5 ppm is

visible, where no unambiguous statement can be made. Since normally, one intense single peak

is obtained, at around 0 ppm with the endgroup at around 15 ppm, partial hydrolysis may be the

reason for this. Peaks from - 10 to - 1 ppm probably arise from degradation products such

as -P-OH and P=O entities. Nevertheless the polymer appeared to be not full degraded yet, since

no distinct phosphate peak at 0.0 ppm is visible 85. However, for clearer statements a lower signal

to noise ratio has to be obtained in future experiments, ideally, already with 31P NMR

measurements taken during the synthesis to follow the process.

PS116.011.esp

70 60 50 40 30 20 10 0 -10 -20 -30 -40 -50 -60 -70 -80 -90

Chemical Shift (ppm)

-0.3

-0.2

-0.1

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Norm

aliz

ed I

nte

nsity

14.5

2 11.7

6

7.8

25.0

62.5

00.9

3

-1.2

4

-2.4

2

-3.8

0-6

.56

-8.7

3

-10.5

0

Figure 28: 31P-NMR spectrum of Polymer 3 in CDCl3.

The aqueous GPC measurement in Figure 28 shows a broad peak at a retention time of about

25.8 min to 27.1 min in all three detectors, refractive index, UV and light scattering. In the refractive

index detector another peak at 30.1 min, emerging from the shoulder of the previous one, is visible.

Pauline Stadler

50

The UV-vis detector was hereby set to an absorbance of 540 nm to detect BODIPY compounds.

Hence, the first peak most probably corresponds to the BODIPY-containing polymer. The second,

smaller compound could be the result of hydrolysis and subsequent degradation of the polymer.

A possible explanation could be the presence of small amounts of water during the

macrosubstitution process or an incomplete macrosubstitution of the Jeffamine within the used

reaction time. Due to the fact, that no signal for the UV-vis detector was detected for the second

peak, most likely no or only small amounts of BODIPY are bound to the degradation products.

Hence, either the BODIPY-compound is cleaved first while the polymer degrades or the amount

of BODIPY is just too small.

Figure 29: Aqueous GPC measurement of Polymer 3.

The UV-vis measurement, depicted in Figure 30, shows an absorbance of 0.5075 for a sample

concentration of 0.2 mg mL-1. With the UV-vis calibration of pentyne-BODIPY for low

concentrations, described in more detail in chapter 4.4.1, the BODIPY-content of the sample could

be calculated. Since the absorbance maximum is the same as for the pentyne-BODIPY at 533 nm,

the calibration should be appropriate. In a 0.2 mg mL-1 concentrated sample in ethanol, a BODIPY-

0 10 20 30 40

0.0

0.2

0.4

0.6

0.8

1.0

norm

aliz

ed d

ete

cto

r re

sponse

retention time / min

Light scattering

UV-vis

Refractive index

25.8 - 27.1

30.1

Pauline Stadler

51

content of 0.0145 · 10-6 mol mL-1 was calculated resulting in a concentration of 3.5 w%. A

theoretical molecular weight of the polymer of 99.7 kDa gives around 7.3 BODIPY molecules per

polymer chain. With the used amount of 3-ethynylaniline-BODIPY a maximum amount of 10

molecules should be on each polymer chain, leading to a conversion of 73 % for this reaction.

Deviations possibly arise due to errors during sample preparation, accuracy of the calibration or

impurities in the sample.

Figure 30: UV-vis spectra of Polymer 3: poly(organo)phosphazene with 3-ethynylaniline-BODIPY and

Jeffamine M-1000 (1:9) sidegroups in ethanol.

The FT-IR spectrum, depicted in Figure 30, shows a very small peak at 1660 cm-1, that may arise

from the triazole ring of the 3-ethynylaniline-BODIPY. Another indication for the presence of the

BODIPY-compound in the product is a peak at 1550 cm-1, stemming from the alkene stretching of

the aromatic BODIPY core. Additional peaks could be assigned as well, where the wavenumber

of 2880 cm-1 corresponds to the alkane C-H stretching, the one at 1240 cm-1 to the phosphazene

stretching and the peak at 1100 cm-1 to the aliphatic ether.

400 500 600 700

0.0

0.2

0.4

0.6

0.8

1.0

533

5335

54

norm

aliz

ed d

ete

cto

r voltage

wavelength / nm

Absorbance

Emission (485 nm)

Excitation (595 nm)

Pauline Stadler

52

Figure 31: FT-IR spectrum of Polymer 3: poly(organo)phosphazene with 3-ethynylaniline-BODIPY and

Jeffamine M-1000.

In summary, it is most likely that Polymer 3 is partially degraded by hydrolysis. The reason may

lie in the presence of water in the reaction mixture or an incomplete macrosubstitution. This would

explain the second peak in the GPC-measurement at smaller molecular weights, as well as the

broad multiplet in the 31P-NMR spectrum. UV-vis absorbance, emission and excitation spectra

show an intact BODIPY-structure, furthermore, it revealed a BODIPY-concentration of 3.5 w%.

For a non-degraded polymer, this would mean that 7.3 BODIPY-molecules are conjugated onto

one polymer-chain, leading to a conversion of 73 %.

4000 3500 3000 2500 2000 1500 1000

0.0

0.2

0.4

0.6

0.8

1.0norm

aliz

ed tra

nsm

itta

nce

wavenumber / cm-1

C-H

stre

tchin

g (a

lkane)

C=

C s

tretc

hin

g (a

lkene)

C=

C s

tretc

hin

g (tria

zole

)

C-O

stre

tchin

g

(alip

hatic

eth

er)

P=

N s

tretc

hin

g (P

hosphazene)

Pauline Stadler

53

4.3 Synthesis of poly(ethylene glycol) monomethyl ether azide

The poly(ethylene glycol) monomethyl ether azide was synthesized through tosylation of

poly(ethylene glycol) monomethyl ether and subsequent azidation according to the reaction

scheme in Figure 32.

The PEG-azide was synthesized in order to use it for a CuAAC with Polymer 2, containing only

propargylamine sidegroups, to enable water solubility. In general, PEG is often referred to as an

ideal compound in biomedical applications due to the fact, that it reduces toxicity and increases

stability against enzymatic degradation 109.

In this synthesis method, the alcohol group is converted into a tosylate that, as a good leaving

group, is substituted by an azide-functionality. In the 1H-NMR of the tosylated PEG [Appendix 9],

a triplet at 3.90 ppm can be assigned to the methylene group next to the tosylate. This triplet shifts

to 3.25 ppm after the azidation [Appendix 10], along with the disappearance of the aromatic

protons of the tosylate.

The azide moiety has been further verified by FT-IR measurement [Appendix 38], as expected an

intense azide stretching peak at 2100 cm-1 can be seen.

Figure 32: Reaction scheme of the tosylation and subsequent azidation of PEG, adapted from literature 99.

Pauline Stadler

54

4.4 Copper-catalyzed azide-alkyne cycloaddition: small molecules

In this type of reaction, the azide functionality of the BODIPY reacts with an alkyne to give a triazole

ring. Four different alkynes were tested for this reaction, where an overview can be seen in

Figure 33. The successful reaction can be verified via 1H-NMR spectroscopy, where the peak at

4.55 ppm, corresponding to the methylene group next to the azide, shifts to about 5.6-5.9 ppm,

depending on the used alkyne. In addition, a peak at 7.50-8.00 ppm arises, originating from the

proton on the double bond of the formed triazole ring.

Figure 33: Overview of small-molecule copper catalyzed azide-alkyne cycloaddition reactions.

Pauline Stadler

55

4.4.1 Synthesis of pentyne-BODIPY

The pentyne-BODIPY was synthesized as a small molecular model compound of the

CuAAC-reaction compared to the coupling onto the polymer.

The synthesis itself could be optimized by adjusting the used equivalence of pentyne from 10 eq.

to 20 eq., obtaining a high yield of 97 %. This considerable excess of pentyne may be necessary

because of the volatility of this compound. Other reactions, employing different alkynes, showed

that normally already a moderate excess of the alkyne of 5 eq. results in high conversions.

The UV-vis absorbance of pentyne-BODIPY looks similar to the other BODIPY-compounds

already described and has its maximum at 533 nm in ethanol. This slightly shifted maximum,

compared to the BODIPY-azide, may come from the formed triazole ring after the CuAAC reaction.

However, since the BODIPY-azide was measured in methanol, this shift could be a result of

solvent effects as well. Due to the fact that the analysis of the amount of BODIPY on the polymer

after click reaction is hard to investigate by other means, a UV-vis absorbance calibration with

pentyne-BODIPY was recorded. The calibration in Figure 34 shows all measured absorbance

values in correlation to their respective concentration. The linear fit of all measurement data shows

some deviations and only reaches a R2-value of 0.989. These differences may occur from dilution

errors, since the concentrations were quite low. Many other reasons for this deviation from the

linearity of Lambert Beer’s law, like pH, temperature, dimer formation, hydrogen bonding, cluster

formation or other molecular interactions, are possible as well. Hence, calibration curves for low

and for high concentrations were established, increasing the R2–value above 0.999 for each and

allowing a more precise determination of the BODIPY content in the respective concentration

ranges. That said, this calibration only gives a rough estimation for the amount of BODIPY on the

polymer, since many effects can influence the absorbance.

Pauline Stadler

56

Figure 34: UV-vis calibration of pentyne-BODIPY with linear fit of the data: red: linear fit of all data,

green: linear fit of low concentrations, blue: linear fit of high concentrations.

4.4.2 Synthesis of BOC-/Fmoc-propargylamine-BODIPY and tested deprotection

methods

As already described above for Polymer 3, chapter 4.2.4, direct macrosubstitution of the BODIPY

compound onto the poly(dichloro)phosphazene precursor would be desirable to allow increased

control over the BODIPY-content. The substitution of the chlorine atoms on the phosphorus

performs reliable and fast 80 and subsequent substitution of the remaining chlorines with

sidegroups like Jeffamine M-1000 could enable water solubility. To this end, a propargylamine-

BODIPY compound should be synthesized bearing a free amine functionality. However, the

CuAAC of propargylamine with the BODIPY-azide according to the general procedure, explained

in chapter 3.6.1, did not lead to the desired product. A possible explanation may be that the amine-

functionality could influence this reaction through interaction with the copper. Therefore, protection

group chemistry was considered. Two protection groups, BOC and Fmoc, were tested, due to their

0.005 0.010 0.015 0.020 0.0250.0

0.2

0.4

0.6

0.8

1.0

Absorb

ance a

t 533 n

m (

Maxim

a)

concentration / 10-6 mol mL-1

experimental data

linear fit: all

linear fit: low concentration

linear fit: high concentration

Calibration

y = 42.81 x - 0.092

R2 = 0.989

y = 37.03 x - 0.030

R2 = 0.999

y = 56.92 x - 0.3642

R2 = 0.999

Pauline Stadler

57

difference in deprotection conditions. The CuAAC of the BODIPY-azide with the protected

propargylamine resulted in the desired product in both cases.

BOC-protected amines are normally deprotected under acidic conditions. A common method

employs trifluoroacetic acid (TFA) in DCM in a ratio of 1:2. Stability tests under these conditions

were performed with the BODIPY-azide leading to decomposition of the BODIPY. According to

literature 110 this may be caused by the influence of Brønsted acids on the BODIPY. TFA could

lead to the removal of the BF2 moiety, therefore destroying the desired compound. The

deprotection method was therefore adapted, according to literature 111, describing a different

BODIPY structure. The utilization of 1 M HCl in 1,4-dioxane did, however, not lead to deprotection

of the amine over the course of 2 hours. Doubling the concentration to 2 M HCl in 1,4-dioxane

resulted again in the destruction of the BODIPY. Iodine-mediated deprotection according to a third

literature procedure 112, where 8-15 mol % of I2 were used in DCM, did not allow for a deprotection,

as well.

With no suitable way of BOC-deprotection Fmoc was investigated as a protecting group, being

normally deprotected under mildly basic conditions. Nevertheless, the results were quite similar.

Deprotection with 5 eq. piperidine and 2 eq. DBU in DCM over 30 minutes lead to destruction of

the molecule. Another literature procedure 113, where 0.2 M aq. NaOH in THF / MeOH (3:1) were

used, did not deprotect the compound at all. The same result was achieved with the usage of

1.2 eq. NaN3 in DMF at 50 °C according to a third literature procedure 114.

In summary, all tested deprotection methods either lead to destruction of the molecule or no

deprotection at all. A theory for this behavior could be that the desired product

propargylamine-BODIPY itself is not stable. The amine functionality may be too close to the boron

center, leading to coordination. As a result, a fluorine anion could be cleaved, as well as a nitrogen

atom from the BODIPY core destroying the compound in the process. A reason for the apparent

increased stability of the protection group under the tested deprotection conditions could be the

proximity to the electron withdrawing BF2 moiety. This behavior is already described for the BOC-

protecting group in literature 115. The previously stated assumption that the amino group could

negatively influence the CuAAC through interactions with the copper could be negated by the

usage of smaller amounts, like 0.1 eq., of copper.

Pauline Stadler

58

4.4.3 Synthesis of 3-ethynylaniline-BODIPY

Building on the theory stated above that the proximity of the primary amine to the boron center

could lead to the destruction of the BODIPY-compound, a different amine bearing alkyne was

tested, namely 3-ethynylaniline.

The synthesis of 3-ethynylaniline-BODIPY was not possible according to the general procedure

for the CuAAC, described in chapter 3.6.1, although a smaller amount of copper sulfate

pentahydrate with 0.05 eq. and (+)-sodium-L-ascorbate with 0.1 eq. was used to avoid interactions

of the copper and the amine functionality. This behavior was already previously described in

literature 55 for aromatic alkynes. The usage of copper(I)iodide in DMF, however, allowed the

synthesis of the desired product with a yield of 32 %.

With this resulting compound a macrosubstitution of the poly(organo)phosphazene precursor

could be done, already described in chapter 4.2.4.

4.5 Copper-catalyzed azide-alkyne cycloaddition: macromolecules

4.5.1 BODIPY-azide on Polymer 1

To obtain a water-soluble BODIPY-containing light-stimulus responsive polymer carrier, the

BODIPY-azide was bound onto Polymer 1, containing Jeffamine M-1000 and propargylamine

substituents (1:1), via a CuAAC of the azide functionality of the BODIPY and the triple bond of the

propargylamine substituent, as demonstrated in Figure 35. This reaction was tested twice with

different reaction conditions, first with a substitution on 50 % of the triple bonds over the course of

24 h resulting in a BODIPY-concentration of 2.7 w%, and second with a substitution of 100 % of

the triple bonds over the course of 3 days resulting in a BODIPY-concentration of 9.6 w%.

Figure 35: Reaction scheme of the copper catalyzed azide-alkyne cycloaddition of the BODIPY-azide and

Polymer 1; with R = Jeffamine M-1000 and propargylamine (1:1) for Polymer 1, n = 50.

Pauline Stadler

59

In general, for both loadings, the 1H-NMR and 31P-NMR measurements showed a successful

synthesis and that the polymer backbone and the sidegroups were still intact after the reaction.

No distinct peaks of the BODIPY could be seen in the proton-NMR, most probably due to its rather

low concentration. In addition, due to the high intensity of the Jeffamine peaks, in general, most

other smaller peaks were covered.

The conjugation of the BODIPY compound onto the polymer was verified via GPC with the UV-vis

detector adjusted to a detector wavelength of 540 nm. This ensured detection of the BODIPY

containing compounds only, since the polymer itself does not absorb at this wavelength.

Estimations regarding the purity could be made as well, as no free BODIPY-azide or other

impurities can be seen at higher retention times.

The amount of BODIPY on the polymer was calculated using the UV-vis calibration of pentyne-

BODIPY. Although quantitative analysis may differ from the actual BODIPY-concentration due to

problems regarding solubility of the products.

Degradation studies and stability tests were performed with Polymer 1, containing 9.6 w%

BODIPY-azide, further described in chapter 4.5.1.2.

In the FT-IR spectra, Figure 36, a very small peak at 1660 cm-1 can be seen for the products,

which may arise from the formed triazole ring. Another new peak, compared to the polymer, at

1573 cm-1 corresponds to the cyclic C=C stretching of the BODIPY core. The azide stretching

peak at 2100 cm-1 is gone, further verifying the absence of free BODIPY-azide in the product.

Pauline Stadler

60

Figure 36: FT-IR spectra of Polymer 1 containing 2.7 w% and 9.6 w% BODIPY compared to the polymer

alone.

4.5.1.1 2.7 w% BODIPY-azide on Polymer 1

In first experiments a lower loading of the polymer was tested to establish the procedure and

subsequent purification. 16 w% of BODIPY were targeted resulting in 2.7 w% in the product.

The aqueous GPC measurement, illustrated in Figure 37, shows an overlap of the detector signals

at 25.5 min, indicating the presence of a lowly disperse polymer without any sideproducts present.

As stated above, the adjusted UV-vis detector wavelength indicates that the BODIPY-compound

is solely present linked onto the polymer. A peak at about 21 min was detected in nearly every

measurement, most probably corresponding to a ghost peak stemming from the column.

4000 3500 3000 2500 2000 1500 1000

0.0

0.2

0.4

0.6

0.8

1.0norm

aliz

ed tra

nsm

itta

nce

wavenumber / cm-1

2.7 w% BODIPY on Polymer 1

9.6 w% BODIPY on Polymer 1

Polymer 1

C=

C s

tretc

hin

g (c

yclic

)C

=C

stre

tchin

g (tria

zole

)

Pauline Stadler

61

Figure 37: Aqueous GPC measurement of Polymer 1, containing 2.7 w% BODIPY with different detector

signals; red: UV-vis detector, green: light scattering detector, blue: refractive index detector.

The UV-vis spectrum, Figure 38, looks similar to previously measured BODIPY compounds. Only

the absorbance spectrum shows a higher peak at about 375 nm, indicating that the BODIPY

structure could be defect or degradation occurred, possibly due to long purification times using

dialysis and vivaspin. Impurities could be a reason for this phenomenon as well.

An absorbance intensity of 0.5071 at the absorbance maximum at 536 nm, comparable to the

pentyne-BODIPY, was detected for a concentration of 0.2 mg mL-1. According to the

pentyne-BODIPY calibration at lower concentrations 0.0145 · 10-6 mol mL-1 BODIPY were

therefore present in the sample, corresponding to a loading of 2.7 w%. Based on the molecular

weight of Polymer 1 around 4 BODIPY molecules were coupled in average per polymer chain.

Compared to the used amount of 0.5 eq. BODIPY, equating on average 25 BODIPY-molecules

per polymer chain, only a conversion of 16 % was achieved.

0 5 10 15 20 25 30 35

0.0

0.2

0.4

0.6

0.8

1.0norm

aliz

ed d

ete

cto

r re

sponse

time / min

UV-vis (540 nm)

Light scattering

Refractive index

25.5 min

Pauline Stadler

62

Figure 38: UV-vis spectrum of Polymer 1, containing 2.7 w% BODIPY in ethanol.

4.5.1.2 9.6 w% BODIPY-azide on Polymer 1

In the following experiment the loading of the BODIPY was tried to be improved. To this end 1 eq.

of BODIPY were reacted with Polymer 1 in an attempt to increase the overall BODIPY content in

the product. Additionally, the reaction time was increased from 1 to 3 days.

The measurement on the aqueous GPC looked similar to the sample with the 2.7 w%

BODIPY-concentration, indicating a successful reaction and high purity.

The UV-vis spectrum in Figure 39 is in agreement to previous recorded data. Compared to the

product with a lower BODIPY-concentration, the peak at about 375 nm is not as high, as described

above an increase at the wavelength could hint at an onset of degradation indicating a still intact

BODIPY molecule for this experiment.

In a 0.086 mg mL-1 concentrated sample in ethanol, an absorbance intensity of 0.8093 at its

maximum of 535 nm was detected. According to the pentyne-BODIPY calibration for higher

350 400 450 500 550 600 650 700 750

0.0

0.2

0.4

0.6

0.8

1.0

533

536

556

norm

aliz

ed d

ete

cto

r voltage

wavelength / nm

Absorbance

Emission (485 nm)

Excitation (595 nm)

Pauline Stadler

63

concentrations around 0.0206 · 10-6 mol mL-1 BODIPY were in the probe, corresponding to

9.6 w%. In contrast to the previous synthesis, 15.5 BODIPY molecules were theoretically linked

onto each polymer chain in average for this attempt. Nevertheless, this only corresponds to a

conversion of 31 %. Since 1 eq. of BODIPY was used for each repeating unit, there should be 50

BODIPY molecules per polymer chain. Still, the increase of the reaction time from 1 to 3 days in

combination with a higher content of BODIPY could increase the conversion of this reaction

by 15 %.

Figure 39: UV-vis spectrum of Polymer 1, containing 9.6 w% BODIPY in ethanol.

Degradation studies, shown in Figure 40, were performed for different irradiation times at an

irradiation wavelength of 365 nm. A monoexponential fitting, represented in Figure 41, was found

for the degradation, slowing down at lower concentrations. At higher concentrations down to

intensities around 0.4 an approximately linear behavior was found. Afterwards, the curve flattens,

leading to an exponential degradation, which was already examined in literature for other

BODIPY-compounds 42. The irradiation wavelength at 365 nm was chosen based on the light

intensity of the available light sources, despite the fact that the absorbance would be greater at

350 400 450 500 550 600 650 700 750

0.0

0.2

0.4

0.6

0.8

1.0532

535

555

norm

aliz

ed d

ete

cto

r voltage

wavelength / nm

Absorbance

Emission (485 nm)

Excitation (595 nm)

Pauline Stadler

64

around 540 nm. The degradation itself can be seen by eye as well as by discoloration of the

irradiated solution from an intense pink/purple to a light rose solution, depicted in Figure 40.

Figure 40: Degradation studie of BODIPY-azide on Polymer 1 with an irradiation wavelength of 365 nm in

ethanol; left: picture of the solution before irradiation; middle: absorbance spectra recorded after certain

irradiation times; right: picture of the solution after 3150 min of irradiation.

Since the degradation in general was found to be rather slow, longer breaks in-between irradiation

times were unavoidable. In Figure 41 the experimental data were corrected for any intensity loss

of the absorbance from storage in the dark.

Pauline Stadler

65

Figure 41: Degradation kinetics of BODIPY-azide bound onto polymer 1, showing an exponential

degradation over time;; experimental data (black spheres), corrected data (red triangles),

exponential fit (solid red line).

To ensure the stability of the BODIPY loaded polymer in the dark stability tests of the compound

were performed. The compound was stored in the dark in ethanol at temperatures between

2 - 10 °C over different time intervals and the absorbance was controlled, the spectra are

represented in Figure 42. An intensity loss of 0.0073 in the absorbance spectrum was observed

for storage overnight and 0.0105 for a storage time of 4 days. This indicates considerable stability

of the compound in the dark even when stored in solution.

0 500 1000 1500 2000 2500 30000.0

0.1

0.2

0.3

0.4

0.5

0.6

0.7 experimental data

corrected data

exponential fit

Ab

so

rban

ce a

t 5

35

nm

/ a

.u.

irradiation time / min

Modell Asymptotic1

Gleichung y = a-b*c^x

Zeichnen C

a 0.14055 ± 0.01105

b -0.59568 ± 0.01149

c 0.99871 ± 6.13387E-5

Chi-Quadr Reduziert 1.19245E-4

R-Quadrat (COD) 0.9976

Kor. R-Quadrat 0.997

Pauline Stadler

66

Figure 42: Stability tests of the BODIPY-azide coupled onto Polymer 1 in the dark in ethanol over different

time periods: overnight (green) and over 4 days (blue).

4.5.2 PEG-azide on Polymer 2

25 % of the triple bonds of the propargylamine sidegroups of the poly(organo)phosphazene

Polymer 2 were used for the CuAAC with PEG-azide to provide water solubility, as shown in the

reaction scheme in Figure 43.

Pauline Stadler

67

Figure 43: Reaction scheme of the copper catalyzed azide-alkyne cycloaddition of the PEG-azide and

Polymer 2.

The reaction was additionally tested with a higher amount of catalyst of 1.5 eq. copper sulfate

pentahydrate and 3 eq. of (+)-sodium-L-ascorbate. However, these conditions did not lead to the

desired product and swelling of the polymer could be observed during the reaction. A possible

explanation for this behavior may be the high amount of copper, forming a complex with the

polymer in the reaction solution. With lower amounts of catalyst, this behavior could be avoided.

The 31P-NMR spectrum of the product, not presented here showed an intact polyphosphazene

backbone without signs of degradation. Due to the fact that the product was water-soluble, the

reaction had been at least partially successful, since the propargylamine substituted

poly(organo)phosphazene was not water soluble at all. In the 1H-NMR only the signals arising

from the PEG were visible since, as for the Jeffamine M-1000, the intensity of these peaks are

quite high.

In the chromatogram of the aqueous GPC a peak at 27 min was visible most probably

corresponding to the adapted poly(organo)phosphazene, yet some smaller impurities at higher

retention times were still present as well. This result was somewhat expected, since no purification

was done after the reaction. A purification procedure still has to be established to avoid high losses

of the product.

The FT-IR spectrum, Figure 44, showed a new peak at 1723 cm-1, that may come from the formed

triazole ring. The azide stretching peak at 2100 cm-1 was not visible anymore, indicating that no

free PEG-azide was present in the product after the CuAAC reaction. The P=N stretching of the

Pauline Stadler

68

polyphosphazene backbone, as well as the C-H stretching of the alkanes could still be seen. The

alkyne C-H stretching peak expectedly decreased because 25 % of those triple bonds should have

been used up for the reaction.

Figure 44: FT-IR spectrum of Polymer 2 adapted with 25% PEG-azide (red) in comparison with the PEG-

azide (blue) and Polymer 2 (green).

4.5.3 Coupling of BODIPY-azide on 25 % PEG-substituted Polymer 2

The Polymer from chapter 4.5.2 was subsequently reacted with BODIPY-azide on the remaining

alkyne functionalities. The resulting product, however partly lost its solubility in water, possibly

because of the hydrophobic BODIPY. Higher amounts of PEG on the polymer could increase the

solubility to obtain the desired properties.

31P-NMR measurements showed no signal even at increased numbers of scans. The

concentration of the polymer was limited by its poor solubility and due to the low content of

phosphorus in the overall macromolecular structure no results could be generated in reasonable

times.

4000 3500 3000 2500 2000 1500 1000

0.0

0.5

1.0

norm

aliz

ed

tra

nsm

itta

nce

wavenumber / cm-1

C-H

stre

tch

ing

(Alk

yn

e)

N-H

stre

tch

ing

(se

co

nd

ary

Am

ine

)

C-H

stre

tch

ing

(Alk

an

e)

N=

N=

N s

tretc

hin

g

P=

N s

tretc

hin

g

C=

C s

tretc

hin

g (tria

zo

le)

Pauline Stadler

69

The aqueous GPC measurement revealed the presence of the BODIPY-compound on the

polymer. However, although purification was done via dialysis, an abundancy of smaller impurities

was present in the product. Purification with a Vivaspin-20 (MWCO = 3 kDa) with an

ethanol / water (1:1) solution did not lead to a pure product, as well.

In the FT-IR spectrum in Figure 45 a new peak at 1646 cm-1 could be seen, that may correspond

to the C=C stretching of the formed triazole ring between the BODIPY and the polymer. This was

slightly shifted to lower wavenumbers compared to the triazole ring between the PEG and the

polymer at 1723 cm-1, described previously for the PEG-substituted Polymer 2. In addition, the

C=C stretching of the cyclic alkene from the BODIPY at 1550 cm-1, already assigned in chapter

4.1.3, was visible too. Since the N-H and the C-H alkyne stretching shifts overlap, no estimations

about the amount of reacted alkyne could be made.

The UV-vis spectrum [Appendix 28] showed the maximum absorbance at a wavelength of 535 nm

(EtOH). A sample concentration of 0.0666 mg mL-1 was leading to an absorbance intensity of

0.8882 resulting in a BODIPY-concentration of 0.022 · 10-6 mol mL-1 according to the pentyne-

BODIPY calibration curve. This corresponds to a content of 12.3 w% BODIPY, approximately 8.2

BODIPY per polymer chain. Since 0.3 eq. of BODIPY were used, 15 BODIPY molecules should

have reacted. Hence, a conversion of 54 % was achieved. Higher conversion compared to the

Jeffamine-polymer was possible, due to less sterical hindrance at the reaction site with PEG

compared to Jeffamine M-1000. In addition, more propargylamine groups were present in the

PEG-substituted poly(organo)phosphazene, leading to statistically more reaction positions.

Nevertheless, the purification process posed a problem since no pure polymer was obtained as in

contrast to the Jeffamine M-1000 polymers conjugated with BODIPY-azide.

Pauline Stadler

70

Figure 45: FT-IR spectrum of the BODIPY-azide on 25 % PEG substituted Polymer 2 (red) in comparison

with the 25 % PEG substituted Polymer 2 (blue).

4.6 Coupling methods testing

The methods to couple a model compound at the meso-position of the BODIPY for subsequent

release were tested with the hydrolysed BODIPY in order to find appropriate reaction conditions.

Two different procedures were conducted according to literature, reaction with an acid chloride 102

and N,N′-dicyclohexylcarbodiimide (DCC) catalyzed coupling with an acid 103, respectively. The

reaction scheme is shown in Figure 46.

4000 3500 3000 2500 2000 1500 1000

0.0

0.2

0.4

0.6

0.8

1.0norm

aliz

ed tra

nsm

itta

nce

wavenumber / cm-1

C-H

stre

tch

ing

(Alk

an

e)

C=

C s

tretc

hin

g (c

yclic

alk

ene)

C=

C s

tretc

hin

g (tria

zo

le fro

m P

EG

)C

=C

stre

tch

ing

(triazo

le fro

m B

OD

IPY

)

P=

N s

tretc

hin

g

C-H

stre

tch

ing

(Alk

yn

e)

N-H

stre

tch

ing

(se

c. A

min

e)

Pauline Stadler

71

Figure 46: Reaction scheme of two different coupling testing methods using hydrolysed BODIPY.

It was found that the reaction with phenoxyacetyl chloride resulted in higher yields than the DCC

coupling with benzoic acid. The reason for this most probably is the high reactivity of the acid

chloride, leading to a yield of 52 % after 4 hours reaction time. The DCC coupling with benzoic

acid is a milder method, where long reaction times of 7 days were used to obtain a yield of 20 %.

The slow progress of the reaction could be caused by the low reactivity of the benzoic acid.

Humidity could influence the reaction progress as well as it could lead to decomposition of the

DCC.

Alongside the general verification by NMR-spectroscopy the ESI-MS spectrum of the hydrolysed

BODIPY coupled with phenoxyacetyl chloride was recorded [Appendix 33] for which the [M + H]+,

as well as the [M + Na]+ peak was found. Another peak corresponds to the [M - F]+ compound,

which was already found for other BODIPY compounds as well.

4.7 Model compound: reaction of the photoreactive OH-group

The methods established for the hydrolysed BODIPY were then used for the reaction with pentyne-

BODIPY, shown in Figure 47, to realize small molecular model compounds suitable for

photochemical characterization of the photocages in general and the photorelease specifically.

Pauline Stadler

72

Figure 47:Reaction scheme of the model compound synthesis using pentyne-BODIPY.

Despite a functioning reaction with phenoxyacetyl chloride with the hydrolysed BODIPY, the

synthesis failed for the pentyne-BODIPY. A possible explanation may be an attack of the acid

chloride on the triazole ring, forming different sideproducts and partly destroying the model

compound. Using a higher amount of acid chloride of 5 eq. served no observable product at all.

Alternatively, DCC coupling with phenylacetic acid resulted in a considerably higher yield of 47 %

over a time period of 22 hours. Reaction conditions were much milder for this reaction as for the

acid chloride, probably being the reason for less side products and an intact compound.

Nevertheless, also the coupling with DCC showed the formation of side products. A BODIPY

compound with the phenylacetic acid, but without the pentyne was observed hinting on low stability

of the triazole ring with the pentyne.

This instability could also be seen in the ESI-MS spectrum [Appendix 34], where the base peak at

a m/z of 451.23 belongs to the [M - pentyne]+ compound, where the pentyne and the formed

triazole ring were cleaved. Only a very small [M + Na]+ peak was visible in this spectrum.

The photorelease studies were carried out in methanol, where the release of phenylacetic acid

takes place according to the reaction scheme in Figure 48. The data were obtained from

measurements at the Department of Chemistry at Masaryk University, Brno, Czech republic.

Figure 48: Reaction scheme of the photorelease of the model compound in methanol.


Pauline Stadler

73

Generally, the quantum yield of fluorescence with a value of (68 ± 5) % was quite high. Hence,

most photons are emitted through fluorescence. The quantum yield of decomposition was

measured in aerated and degassed solution, shown in Figure 49, resulting in ɸ𝑑𝑒𝑐

of 4.6 x 10-5 %

for the aerated and 7.8 x 10-5 % for the degassed sample. This value is relatively low, leading to

a slow decomposition and release of the cargo. To improve these photorelease properties, either

the leaving group has to be optimized or the framework of the BODIPY has to be altered.

Introduction of heavy atoms at the 2,6-position, as well as boron alkylation is known to improve

the quantum yields of release 54.

Figure 49: Kinetic study of the photoreaction of the BODIPY model compound in aerated methanol

solution (4 · 10-5 M) (left) and in degassed methanol solution (6 · 10-5 M) (right) upon irradiation at 505 nm

for the indicated time span.

The release of the leaving group phenylacetic acid was quantified via HPLC. The measurements,

seen in Figure 50, showed a release of the phenylacetic acid for the irradiated sample at a

retention time of approximately 8 minutes. The yield of the leaving group for the degassed sample

was with a value of (68 ± 4) % slightly higher compared to the aerated sample with a yield of

(65 ± 3) %.

Pauline Stadler

74

Figure 50: HPLC-measurements of the kinetic study of the photoreaction of the BODIPY-azide model

compound showing release of the leaving group phenyl acetic acid (indicated in the red circle).

4.8 Reaction of BODIPY-azide with phenylacetic acid

The reaction was carried out in order to use the product for a CuAAC with Polymer 1, resulting in

a phenylacetic acid loaded polymer, allowing testing of the photorelease from the polymer.

The DCC coupling of the BODIPY-azide with phenylacetic acid resulted in a yield of 69 % after a

reaction time of 18 hours, relatively high, compared to previously described yields of this coupling

reaction with pentyne-BODIPY.

In the ESI-MS spectrum [Appendix 35] the basepeak was identified as the [M - N3]+ species. This

cleavage of the azide moiety was already described for the BODIPY-azide above.

Pauline Stadler

75

4.9 Loading on polymer

4.9.1 Coupling of 9.6 w% BODIPY on Polymer 1 with phenylacetic acid

Loading with phenylacetic acid was performed on a BODIPY-containing polymer via DCC

coupling. With this method a high excess of phenylacetic acid could be used, since it was

commercially available. Despite that, it could not be ensured that after the reaction every BODIPY

was loaded with the reagent successfully and analytics of the bound phenylacetic acid were

challenging.

The aqueous GPC-measurement revealed that the retention time of the polymer peak stayed at

25.5 min, as expected. To detect the phenylacetic acid, a UV-vis absorbance spectrum was

recorded with a wavelength down to 190 nm. An intense peak at about 200 – 230 nm was

detected, corresponding to the phenylacetic acid. Despite that, no quantitative analytics could be

made, since the polymer and the formed triazole ring absorb in this region as well, making a

calibration with phenylacetic acid not applicable in this case. To overcome this drawback, other

compounds with different absorbance maxima would have to be tried to ensure loading on the

polymer with DCC and establish the loading efficiency. The only clear assumption that could be

made was that the polymer was stable under the used coupling conditions.

4.9.2 CuAAC of phenylacetic acid-BODIPY-azide with Polymer 1

To ensure complete loading of the BODIPY molecules on the polymer, the

phenylacetic acid-BODIPY-azide, described in chapter 4.8, was used for the CuAAC with

Polymer 1. In addition, indirect quantification of the phenylacetic acid could be done via

quantification of the BODIPY compound.

The aqueous GPC measurement showed a single peak at an elution time of 25.5 min, in

agreement with previous measurements of Polymer 1 indicating the successful loading of the

phenylacetic acid bearing BODIPY onto the polymer.

Due to solubility problems, the quantification of the BODIPY amount on the polymer was done in

a 0.6 M aqueous HCl solution. The product was dissolved in 6 M HCl and further diluted. The

UV-vis absorbance spectrum showed a maximum intensity of 0.7863 at a wavelength of 522 nm

for a sample concentration of 0.1 mg mL-1. With this value an amount of 9.9 w% phenylacetic acid-

BODIPY could be calculated using the UV-vis calibration of pentyne-BODIPY for high

concentrations. This revealed a content of 12.3 BODIPY-molecules per polymer chain, leading to

Pauline Stadler

76

a conversion of 32.8 %, comparable to the conversion of the reaction without coupled phenylacetic

acid. However, this is only a rough estimation due to the fact that the maximum absorbance

wavelength differs from the one of pentyne-BODIPY, at 535 nm. In addition, the HCl may influence

the absorbance, shifting the absorbance maximum from 524 nm to 522 nm and the baseline was

higher than for previous measurements. Hence, the product was also dissolved in ethanol,

although some undissolved particles were still present. The UV-vis absorbance measurement,

shown in Figure 51, resulted in a slightly lower maximum intensity, explainable by some

undissolved particles. Emission and excitation spectra are in agreement to preliminary data. No

quantification of the phenylacetic acid was possible, since there was an overlap in the UV-vis

absorbance spectra, as discussed before in chapter 4.9.1.

Figure 51: UV-vis spectrum of phenylacetic acid-BODIPY on Polymer 1 in ethanol.

400 500 600 700

0.0

0.2

0.4

0.6

0.8

1.0

524

519

544

norm

aliz

ed d

ete

cto

r voltage

wavelength / nm

Absorbance

Emission (485 nm)

Excitation (595 nm)

Pauline Stadler

77

4.10 Comparison of BODIPY-Polymer coupling methods

As discussed above in different chapters different coupling methods were investigated to

covalently bind a BODIPY-azide compound on a polymer. The methods and their results are

summarized in Table 3, where one method included direct macrosubstitution on the polymer

precursor during polymer synthesis and another employed a BODIPY-azide already loaded with

phenylacetic acid as model drug. The direct macrosubstitution resulted in a high conversion of

73 %, since the chlorine sidegroups can be easily substituted, as described already above.

However, characterization of this product revealed that partial degradation may have occurred. In

another BODIPY-Polymer coupling technique the BODIPY-azide or the BODIPY-azide already

loaded with phenylacetic acid was reacted via a CuAAC with Polymer 1. For these reactions,

conversions of 31-33 % were achieved within a reaction time of 3 days. A decrease in reaction

time resulted in a significant decrease of conversion, where only 16 % within 1 day were reached.

The conversion was generally much lower, compared to the macrosubstitution. Most probably,

sterical hindrance of the Jeffamine groups can prohibit and slow down the reaction. Building on

this in the last tested method, a different polymer was considered, the 25 %-PEG substituted

Polymer 2. A higher conversion with 54 %, compared to the reaction with Polymer 1 was achieved

for the same reaction times, strengthening the theory regarding the availability of the reaction sites

and sterical hindrance due to the Jeffamine. However, the aqueous GPC showed numerous

impurities, even after thorough purification. All in all, macrosubstitution of the

poly(dichloro)phosphazene was the most efficient BODIPY-Polymer coupling technique, but

required an amine-functionalized BODIPY-azide, which was hard to obtain in high yields. In

addition, a synthesis procedure to obtain a stable, water-soluble product still has to be established.

The efficiency of the CuAAC as a BODIPY-Polymer coupling method has to be improved to obtain

reasonable yields, where most probably sterical hindrance has to be minimized.

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Table 3: Comparison of synthesized BODIPY-containing polymers in regards of their efficiency;

N(BODIPY) = BODIPY-molecules per polymer chain.

compound

used

amount of

BODIPY

w% max.

w% N(BODIPY)

max.

N(BODIPY) conversion

Polymer 3 0.2 eq. 3.5 5.3 7.3 10 73 %

BODIPY-azide on Polymer 1 0.5 eq. 2.7 17 4 25 16 %

BODIPY-azide on Polymer 1 1 eq. 9.6 34 15.5 50 31 %

BODIPY-azide on PEG-

substituted Polymer 2 0.3 eq. 12.3 25 8.2 15 54 %

Phenylacetic acid loaded

BODIPY-azide on Polymer 1 0.75 eq. 9.9 33.6 12.3 37.5 33 %

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5 Conclusion and outlook

To sum up, this master thesis presents the synthesis of a novel green-light photoresponsive

heterobifunctional meso-methyl BODIPY derivative and its attachment onto a

poly(organo)phosphazene, as well as the synthesis of a small molecular model compound for its

photophysical characterization and photorelease studies of a model cargo.

Starting with the synthesis of a simple BODIPY framework structure, previously described in

literature, a heterobifunctional azide-containing BODIPY could be obtained after a few synthesis

steps, including hydrolysis of the acetyl group on the meso-position, bromination and subsequent

azidation on either the C3 or C5 position. During this work, the yields of those steps could be

significantly enhanced through changing various reaction conditions. Detailed characterization by

1H-NMR, HSQC, UV-vis, FT-IR and X-Ray revealed the desired structure could be attained in high

purity, however low yields of the last synthesis step, only up to 29 %, limited its use for further

experiments.

To obtain a small molecule model compound, pentyne-BODIPY was synthesized via a copper

catalyzed azide-alkyne cycloaddition, making use of the azide functionality of the BODIPY-azide

and the triple bond of the pentyne, forming a triazole ring in between both molecules. As expected

for click reactions, high yields up to 97 % could be achieved. For photorelease studies,

phenylacetic acid was bound to the meso-position of the pentyne-BODIPY via DCC coupling.

Photorelease studies gave high quantum yields of fluorescence of 68 ± 5 %, but quite low quantum

yields of decomposition with values of 4.6 x 10-5 and 7.8 x 10-5 in aerated and degassed methanol

solutions, respectively. The yield of the released leaving group, namely phenylacetic acid, was

determined via HPLC resulting in a yield of (65 ± 3) % and (68 ± 4) % in aerated and degassed

methanol solutions, respectively. The release kinetics could be improved in future experiments by

changing to a better leaving group, for example, to a carbonate. Alternatively, alterations to the

BODIPY network could be undertaken, for example, by boron alkylation or the introduction of

heavy atoms at the 2,6-positions of the BODIPY to stabilize the triplet state.

For the attachment of the BODIPY onto a polymer, poly(organo)phosphazenes were synthesized

due to their numerous advantages for biomedical applications. Different substituents on the

poly(organo)phosphazene were chosen to achieve the desired properties of the polymer, namely

Jeffamine M-1000 or poly(ethylene glycol) for water-solubility and propargylamine as reactive

moiety for further functionalization. Two different polymers were synthesized, where Polymer 1

contains a mixture of Jeffamine and propargylamine, while Polymer 2 only contains the reactive

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substituent, which was further substituted with PEG-azide. A successful synthesis of the polymers

was proven by 1H-NMR, 31P-NMR, FT-IR and GPC measurements. The BODIPY-azide compound

was then coupled to the polyphosphazene via CuAAC chemistry. For the reaction with Polymer 1,

a BODIPY concentration of 9.6 w%, an average of 15.5 BODIPY molecules per polymer chain,

could be achieved, resulting in a conversion of 31 %. Photocleavage studies showed

monoexponential degradation in ethanol at an irradiation wavelength of 365 nm. The reaction with

the PEG substituted Polymer 2 enabled a higher conversion of 51 %. Most probably, sterical

hindrance and availability of reaction sites were responsible for this result. However,

characterization of the product showed that degradation might have occurred due to uncompleted

macrosubsitution or presence of water during the reaction. The verification of a successful

coupling reaction was performed on an aqueous GPC with the UV-vis detector adjusted to a

detector wavelength of 540 nm, detecting the BODIPY-compounds on the polymer. An alternative

BODIPY-Polymer coupling method was also tested, whereby a BODIPY-compound bearing a free

amine moiety was used directly for the macrosubstitution of the poly(dichloro)phosphazene

precursor, since it is known that this substitution proceeds fast and highly efficient. For this, the

BODIPY-azide was reacted with an amine-functionalized alkyne, namely 3-ethynylaniline. The

reaction was also tested with propargylamine as an alkyne, but results indicated that this

compound may be unstable. Polymer 3 was synthesized with 3-ethynylaniline-BODIPY and

Jeffamine (1:9), but 31P-NMR and GPC-measurements showed partial destruction of the polymer,

possibly explained by incomplete macrosubstitution or presence of water during the reaction.

Nevertheless, the UV-vis and the FT-IR measurements indicate an intact BODIPY structure,

expectedly obtaining a high conversion of 73 % according to the UV-vis calibration.

Loading of the polymer with the model cargo phenylacetic acid was done on one hand via DCC

coupling of phenylacetic acid on the 9.6 w% BODIPY-containing Polymer 1 and on the other hand

via a CuAAC with a phenylacetic acid loaded BODIPY-azide. Since loading after the BODIPY-

Polymer coupling cannot ensure complete loading on each BODIPY and quantification of the

phenylacetic acid was not possible, the BODIPY-Polymer coupling using an already loaded

BODIPY was regarded as the superior approach.

Results, described in this thesis serve as a proof of principle for the design and synthesis of a

macromolecular photocage. Future work has to be done to investigate the improvement of the

release kinetics by change of the leaving group or modification of the BODIPY network. Such

materials are of considerable interest, for example for the light triggered release of drugs and thus

could be investigated with different therapeutic agents.

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113. Přibylka, A., Krchňák, V. & Schütznerová, E. Environmentally Friendly SPPS II: Scope of Green Fmoc Removal Protocol Using NaOH and Its Application for Synthesis of Commercial Drug Triptorelin. J. Org. Chem. 85, 8798–8811 (2020).

114. Chen, C. C. et al. A mild removal of Fmoc group using sodium azide. Amino Acids 46, 367–374 (2014)

115. Smithen, D. A. et al. Use of F-BODIPYs as a protection strategy for dipyrrins: Optimization of BF 2 removal. J. Org. Chem. 77, 3439–3453 (2012).

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Appendix

Appendix 1: 1H-NMR of the BODIPY-framework.

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Appendix 2: 1H-NMR of the hydrolysed BODIPY.

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Appendix 3: 1H-NMR of the BODIPY-azide.

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Appendix 4: HSQC-NMR of BODIPY-azide.

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Appendix 5: 1H-NMR of Polymer 1.

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Appendix 6: 1H-NMR of Polymer 2

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Appendix 7: 31P-NMR of Polymer 1.

Appendix 8: 31P-NMR of Polymer 2.

PS30-01_31P.001.esp

64 56 48 40 32 24 16 8 0 -8 -16 -24 -32 -40 -48 -56 -64 -72


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PS071_31P.010.esp

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Appendix 9: 1H-NMR of poly(ethylene glycol) monomethyl ether tosylate (PEG-tosylate).

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Appendix 10: 1H-NMR of poly(ethylene glycol) monomethyl ether azide.

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Appendix 11: 1H-NMR of BOC-propargylamine.

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Appendix 12: 1H-NMR of Fmoc-propargylamine.

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Appendix 13: 1H-NMR of pentyne-BODIPY

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Appendix 14: HSQC-NMR of pentyne-BODIPY

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Appendix 15: 1H-NMR of BOC-propargylamine-BODIPY

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Appendix 16: 1H-NMR of Fmoc-propargylamine-BODIPY

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Appendix 17: 1H-NMR of 3-ethynylaniline-BODIPY.

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Appendix 18: 1H-NMR of hydrolysed BODIPY + phenoxyacetyl chloride.

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Appendix 19: 1H-NMR of hydrolysed BODIPY + benzoic acid.

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Appendix 20: 1H-NMR of pentyne-BODIPY + pheoxyacetyl chloride.

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Appendix 21: 1H-NMR of pentyne-BODIPY + phenylacetic acid (model compound).

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Appendix 22: HSQC-NMR of pentyne-BODIPY + phenylacetic acid (model compound).

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Appendix 23: 1H-NMR of BODIPY-azide + phenylacetic acid.

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Appendix 24: HSQC-NMR of BODIPY-azide + phenylacetic acid.

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Appendix 25: UV-vis spectrum of the hydrolysed BODIPY.

Appendix 26: UV-vis spectrum of the BODIPY-azide.

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Appendix 27: UV-vis spectrum of pentyne-BODIPY + phenylacetic acid (model compound).

Appendix 28: UV-vis spectrum of 12.3 w% BODIPY-azide on 25 % PEG-substituted Polymer 2.

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Appendix 29: ESI-MS spectrum of the BODIPY-framework.

Appendix 30: Isotope pattern of the [M+H]+ peak of the BODIPY-framework.

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Appendix 31: ESI-MS spectrum of the hydrolysed BODIPY.

Appendix 32: ESI-MS spectrum of the BODIPY-azide.

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Appendix 33: ESI-MS spectrum of hydrolysed BODIPY with phenoxyacetyl chloride.

Appendix 34: ESI-MS spectrum of pentyne-BODIPY + phenylacetic acid (model compound).

Appendix 35: ESI-MS spectrum of BODIPY-azide + phenylacetic acid.

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Appendix 36: FT-IR spectrum of Polymer 1.

Appendix 37: FT-IR spectrum of the Polymer 2.

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am

ine

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ho

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C-H

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N-H

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ary

Am

ine

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CΞ

C s

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hin

g (A

lkyn

e)

P=

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tretc

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Appendix 38: FT-IR spectum of poly(ethylene glycol) monomethyl ether azide.

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