Image 04 : Gauche : Vésicules de type hybride polymère-amphiphile. Droite : Vésicule fluorocarbonée dans un micro-compartiment de polymersome géant. Enfin, des vésicules fluorocarbonées incorporant dans la membrane des senseurs de Ca 2+ photolabiles d'une part et des senseurs moléculaires fluorescents d'autre part, ont été encapsulées dans des polymersomes géants. Ce type de nanostructure multicompartimentalisée a ensuite été irradié, induisant 1) la libération des ions Ca 2+ complexés aux vésicules de type éjecteur, puis 2) leur transfert vers les vésicules de type récepteur fluorescent. Aussi, une augmentation de fluorescence a été enregistrée. La cinétique de libération des ions Ca 2+ a également été déterminée, par suivi de la vitesse d'augmentation de fluorescence. La cinétique observée s'est avérée du même ordre que la cinétique de décomplexation observée pour les molécules de type DMNPE en solution. Le dernier chapitre s´est focalisé sur l'approche de multi-compartimentalisation en utilisant des vésicules fluorées incorporant des fluorophores émettant dans le bleu, le vert ou le rouge. En suivant la même procédure d'encapsulation développée dans les chapitres précédents, des microstructures émettant la lumière blanche ont été obtenues. Toutefois, si ces objets sont observés par microscopie confocale, tout compartiment portant de vésicules de couleur différente a pu être observé, comme imagé en image 05. Image 05 : à gauche : polymersome multi-compartimentalisé émettant la lumière blanche observé par microscopie confocale ; à droite: le même polymersome observé par microscopie pseudo-épifluorescente. En résumé, le but de ce projet de thèse visait au développement du transfert d'ions photoinduit entre compartiments confinés. Nous avons montré avec succès la préparation de tels systèmes, en développant des vesicules fluorées encapsulées dans polymersomes géants. Ces systèmes multicompartimentalisés ont ensuite été exploités pour des études de transfert d'ions, mais aussi pour l'obtention de la lumière blanche en milieu confiné. De plus, nous avons également développé un nouveau fluorophore de type BODIPY et montré son utilisation potentielle pour des applications dans la fonctionnalisation de surface ou le marquage de protéines.

Chapter 3 introduces fluorescent Ca 2+ switches and decaging agents, their synthesis and spectroscopic characterisation. Photoinduced electron transfer rates for hydro soluble Ca 2+sensors are determined, ion complexation studies for amphiphilic and water soluble sensors will be described and Ca 2+ -transfer under pseudo-physiological conditions are shown.

Chapter 4 focuses on the preparation of self-assembled synthetic vesicles with the capability to sense Ca 2+ through the incorporation of amphiphilic Ca 2+ -sensors prepared in chapter 3 in their bilayer. Similarly, preparation of Ca 2+ -decaging vesicles is described. Ion transfer between these self-assembled nanoobjects is studied via fluorescence spectroscopy. As a final approach towards the goal of the project the ion transfer between vesicles multicompartmentalized in the interior of giant polymersomes will be shown via confocal microscopy.

Chapter 5 takes the multicompartmentalization approach one step further on incorporating three different types of orthogonal emissive vesicles in the lumen of a giant polymersome to create white light-emitting microcompartments.

want to respectfully thank him for nourishment and feasts in his place and for the Belgium beer night, but we don't talk about Belgium beer night.

During this multidisciplinary thesis I had the pleasure to collaborate with the group of Prof.

Sébastien Lecommandoux at the LCPO and Dr. Reiko Oda at the IECB. I want to thank both collaboration partners for their contribution and the fruitful discussion concerning the work, but above all for the patience when I asked for the third time: "Sorry, but can you explain that again". I want to thank in particular my invited jury members namely Olivier Sénèque, Christopher Hunter and Ludovic Jullien for taking the time to evaluate my work and for the suggestions to improve the quality of my work.

My deep gratitude goes in particular to the founding bodies of the project. This Ph.D. thesis was financed by the ERC (FP7/2007-2013) "Ideas" program, grant agreement N°208702. All the work presented in this thesis corresponds to and directly contributed to the objectives of the project ("COMMOTION"). Equally, support from the initiative d'excellence at Bordeaux via a project "Projet exploratoire premier soutien" is greatly appreciated. I also want to thank the people who directly contributed to this work namely Dr. Julie Thevenot for excellent confocal microscopy experiments along with Guillaume Raffy who are in my eyes magicians on the instrument. Guillaume Vives, Pinar Batat and Gedminas Jonusauskas are greatfully acknowledged for the participation with precursors and transient absorption measurements and Jean-Marc Vincent for his guidance on some ultrafast catalysts.

The CESAMO group, who helped me with fluorine NMR samples and mass spectroscopy, is highly acknowledged. I want to thank Jean-Michel, Noël, Claire, Patricia and the rest of the team especially for the last few weeks before I had to hand in this manuscript and I came only with very urgent samples to measure…. and they did.

A big thanks goes to the people who made my everdaylife in the lab a pleasant experience.

During the three years I conducted my thesis I had the pleasure to meet many people from all over the world. So thanks to HP, Arnaud, Aurélien, Peter Verwilst, Luca, Cedric, Quentin and Sergey for making life in the lab despite crazy hours quite lively. I want to thank as well my combatants in other labs with whom I spend my leasure time when there was some. Suresh, Kitkat, Salem and the rest of the NSysA doctorants and postdocs for the good times we had.

Natürlich darf ich die Leute in Deutschland nicht vergessen die ich während dieser drei Jahre ziemlich vernachlässigt habe, allen voran meine Familie die mich maximal zweimal im Jahr zu sehen bekam. Ganz besonders möchte ich auch meinen Freunden danken die in dieser Zeit so gut wie gar nichts von mir hörten. Einen ganz speziellen Dank natürlich an Alex der mir das Gefühl gab, auch wenn ich mich mal wieder ewig nicht gemeldet hatte, dass wir uns erst letze Woche das letzte mal gesehen haben. So finally I kept the last acknowledgements for my hearts person, my adorable girlfriend Léa Messager. I want to thank her especially for her love and support in the last few months and for an awesome time together during these three years.

The final line is reserved for all the people I forgot to mention because I was very late for preparing my manuscript and there might be the chance that some people who deserved to me mentioned here, didn't find the way on these pages. So a big thank you to them for whatever they did.

Résumé de thèse :

Communication moléculaire biomimétique entre molécules et nanosystèmes photocontrôlés en compartiments confinés.

Robin Bofinger

L'objectif de cette thèse est l'étude du transfert des ions entre nanocompartiments confinés afin de mimer la communication cellulaire des systèmes biologiques. Pour cette raison, nous avons envisagé une approche multidisciplinaire combinant une gamme de techniques dans différents domaines de chimie tels que la chimie supramoléculaire, la chimie des matériaux et la photochimie (utilisation de molécules photoactives). Après une introduction du sujet de thèse et une étude bibliographique des domaines de chimie utilisés, les résultats accomplis se présentent en quatre chapitres.

Le premier chapitre de résultats se focalise sur la synthèse et l'étude d'un nouveau fluorophore à base de BODIPY (bore-dipyrométhene) portant un cycle aromatique perfluoré.

La photostabilité de ce fluorophore s'est révélée trois fois supérieure à celle de la fluorescéine, utilisée comme standard pour la microscopie de fluorescence.

Dans un premier temps, le cycle perfluoré a montré une bonne réactivité avec les amines et les thiols, groupements fonctionnels d'intérêt biologique. De plus, une réaction simultanée de ce fluorophore avec des amines et des thiols en solution montrait une conversion sélective de 99 % en produit thiolé.

Abbreviations:

Chemicals and bio molecules:

General introduction

The cell is the ultimate supramolecular system harnessing a wide range of supramolecular interactions such as ion-dipole, dipole-dipole, hydrophobic interactions or hydrogen bonding.

A major area of supramolecular chemistry is dedicated to reproducing or mimicking certain aspects of these highly sophisticated architectures. Progress in the molecular biology domain allows a deeper understanding and gives access to a number of natural processes which gave rise to some machines exploiting the concepts nature is using for billions of years. Light is a environmental stimulus which is involved in natural processes such as photosynthesis or the mechanism of vision in the human eye. A part of supramolecular photochemistry is therefore dedicated to the exploitation of light as trigger for molecular processes and interactions. In order to control and fine tune biological processes in a cell-like activation of enzymes when needed or triggering of adhesion to other cells, the nanocompartments inside of the cell need to communicate with one another. For this type of cellular communication a messenger is needed and among others the divalent cation Ca 2+ emerged as one of the most ubiquitous ones. The goal of this thesis is the mimicry of this Ca 2+ communication in rudimentary artificial systems by using the environmental stimulus light as a trigger to control the process.

Advances in material science and self-assembly made it possible to create capsules which can resemble the membrane of cells. The progress in supramolecular chemistry gave us the understanding and the means to mimic a set of intracellular interactions, in particular between ions and receptors to create molecules which bind and release ions on command. Advances in supramolecular photochemistry yielded the tools to detect the presence and absence of ions via fluorescence reporters. The multidisciplinary approach of this project will make use of the aforementioned domains of chemistry to show an approach to the light controlled mimicry of cellular communication in artificial systems.

Chapter 1 of this thesis gives an introduction to supramolecular chemistry and highlights the importance of ions in biology. The underlying principles of photochemistry and fluorescent reporter molecules are also given.

Chapter 2 deals with the synthesis and characterisation of a novel boron-dipyrromethene (BODIPY) fluorophore and its application as a selective label, single molecule emitter and for surface patterning 1 Introduction

Ions in biology

Many ions play a vital role in biological systems. Among the most common ions one can find H + , Na + , K + , Ca 2+ , Mg 2+ , Cl -, PO 4 3-, HCO 3 -, and OH -. These ions play very different roles in cells or the human body. While Na + is kept at rather low concentrations, K + is the major counterion in the interior of a cell, therefore available in concentrations far higher than the other ions (table 01). The regulation of the ions is conducted by the Na + /K + -ATPase which exchanges 3 sodium ions from the interior of the cell for 2 potassium ions, creating a charge difference which is one of the mechanisms to keep the resting membrane potential of the cell. Mg 2+ is second most abundant cation in the eukaryotic cell 3 and the major counterion of adenosine triphosphate (ATP), the so called fuel of the cells and other nucleic acids 4 . Indeed, when one talks about ATP generally this refers to the magnesium salt of ATP. Besides that, Mg 2+ controls various enzymes and is part of cofactors. The most interesting ion for our studies is, however, Ca 2+ , which is the richest element in the human body. This is mainly due to the presence in human bones. The reason we are interested in Ca 2+ is however for its role in the signal transduction inside of eukaryotic cells. Several second messengers are known nowadays, with the complementary system cyclic adenosine monophosphate (cAMP) and Ca 2+ among the most important ones. 5 The crucial role of cAMP was discovered by Sutherland and Rall 6 who identified the molecule as a mediator for the hormones glucagon and ephinedrine in the glycogen metabolism in the liver. Not very long after the discovery of cAMP as a second messenger, Ca 2+ was established by Heilbrunn 7 even though the importance of Ca 2+ in the event of muscle contraction had been shown much earlier by Ringer 8 . Nowadays, it is known that Ca 2+ plays an essential role in almost all biological processes and is considered the universal messenger in biological systems. The free Ca 2+ concentration in the cytosol is maintained rather low (10 -4 mM) due to Ca 2+ -ATPases located in the cell wall and endoplasmic reticulum 9 . Keeping a low concentration sets the stage for intracellular signal transduction. An increase in the intracellular Ca 2+ -level is mostly achieved by voltage-guided Ca 2+ -pumps which can increase the intracellular concentration >10-fold in milliseconds. In neurons the Ca 2+ increase can be even faster. 10 One of the most prominent proteins which react to changes in Ca 2+ -concentration is calmodulin (see figure 01), containing 4 high affinity binding sites. In the absence of Ca 2+ the helices of calmodulin are packed so that their hydrophic part is not exposed to the exterior. Upon binding Ca 2+ the protein undergoes a conformational change so that the hydrophic helices are exposed and other enzymes or proteins can bind. 11 CaM structure (c) CaM:smMLCK peptide complex structure. Each structure is shown as a cartoon and spacefilling model. 12 This is just one of many examples where Ca 2+ triggers a cellular event. Others would include the release of neurotransmitters 13,14 or the involvement in programmed cell death. 15 Considering the universal application of Ca 2+ as messenger in biological systems, the adaption of the Ca 2+ based communication for artificial systems is a self-evident approach.

Goal of the project

In order to study ion transfer in artificial systems one can divide the process into three events.

First event is the ion binding by a receptor molecule that, after receiving a stimulus, will eject the ion into the media. For this work light was chosen as the ion-ejecting stimulus. The second event is ion transfer or diffusion and finally the arrival of the ion and the detection by an appropriate reporter. Ion detection by reporter molecules can be achieved by a structural change or a change in physico-chemical properties.

In this thesis a photo-guided approach to biomimetic cellular communication in artificial systems is considered. Therefore Ca 2+ -based communication will be established between vesicles representing Ca 2+ storages in eukaryotic cells, in the interior of µm-scale giant polymersomes, which will represent the cell. Controlling this event by light will offer a realtime control over the ion transfer occurring inside the artificial cell mimicry. Detection of the ion is facilitated by fluorescent-switches as reporter molecules in the membrane of a second nanoobject, representing the receiving macromolecule inside the cell.

Supramolecular chemistry

Supramolecular chemistry was defined by one of its pioneers, Jean as the "chemistry beyond the molecule".

concerns the non-covalent bonds between host molecules and guest molecules. E supramolecular chemistry using artificial systems is a young discipline having been launched

in the 1970's, nature itself uses by definition supramolecular assemblies to operate since the beginning of life. In biological chemistry the supramolec receptor sites of enzymes and the guest molecules are ligands, ions, cofactors or drugs. Most of these interactions rely on supramolecular interactions such as ion or hydrophobic interactions. In th lipid bilayer, therefore as mentioned in chapter 1, Na transport ions actively or passively over the lipophilic barrier. Another mechanism for the transport of K + ions over the mytochondria membrane is via the carrier mechanism. In this case, valinomycin wraps itself, due to creating a hydrophobic supermolecule which can traverse the lipid bilayer.

structure and the molecular model are presented in figure 03. Beside the interactions mentioned, there are several other ways that molecules can interact with each other, without forming a real covalent bond. A way to quantify these interactions has been proposes in the group of Hunter supramolecular binding. A list of non table 02. An important consideration for these interactions with regard to guest binding is the competitive nature of the medium. Indeed, highly polar protic strength found in Nature are typically not conducive with effective binding. These factors are considered below in guiding the choice of the calcium receptor.

8 Supramolecular chemistry pramolecular chemistry was defined by one of its pioneers, Jean-Marie Lehn, who as the "chemistry beyond the molecule". 16 In its simplest form, supramolecular chemistry covalent bonds between host molecules and guest molecules. E supramolecular chemistry using artificial systems is a young discipline having been launched

in the 1970's, nature itself uses by definition supramolecular assemblies to operate since the beginning of life. In biological chemistry the supramolecular hosts are, for example, the receptor sites of enzymes and the guest molecules are ligands, ions, cofactors or drugs. Most of these interactions rely on supramolecular interactions such as ion-dipole, hydrogen bonding or hydrophobic interactions. In the cell for example, cations can not pass freely through the lipid bilayer, therefore as mentioned in chapter 1, Na + /K + -ATPase and other features need to transport ions actively or passively over the lipophilic barrier. Another mechanism for the ions over the mytochondria membrane is via the carrier mechanism. In this case, valinomycin wraps itself, due to ion-dipole interactions, around the potassium ion creating a hydrophobic supermolecule which can traverse the lipid bilayer.

structure and the molecular model are presented in figure 03.

left: Valinomycin molecular model binding a potassium ion, right: chemical structure of he interactions mentioned, there are several other ways that molecules can interact with each other, without forming a real covalent bond. A way to quantify these interactions has been proposes in the group of Hunter 19 which can be applied to almost any ty A list of non-covalent interactions and their strengthes is given in table 02. An important consideration for these interactions with regard to guest binding is the competitive nature of the medium. Indeed, highly polar protic solvents and elevated ionic strength found in Nature are typically not conducive with effective binding. These factors are considered below in guiding the choice of the calcium receptor.

Marie Lehn, who coined it

In its simplest form, supramolecular chemistry covalent bonds between host molecules and guest molecules. Even though supramolecular chemistry using artificial systems is a young discipline having been launched

in the 1970's, nature itself uses by definition supramolecular assemblies to operate since the ular hosts are, for example, the receptor sites of enzymes and the guest molecules are ligands, ions, cofactors or drugs. Most dipole, hydrogen bonding e cell for example, cations can not pass freely through the ATPase and other features need to transport ions actively or passively over the lipophilic barrier. Another mechanism for the ions over the mytochondria membrane is via the carrier mechanism. In this , around the potassium ion creating a hydrophobic supermolecule which can traverse the lipid bilayer. 17,18 The molecular left: Valinomycin molecular model binding a potassium ion, right: chemical structure of he interactions mentioned, there are several other ways that molecules can interact with each other, without forming a real covalent bond. A way to quantify these interactions which can be applied to almost any type of covalent interactions and their strengthes is given in table 02. An important consideration for these interactions with regard to guest binding is the solvents and elevated ionic strength found in Nature are typically not conducive with effective binding. These factors are The part of supramolecular chemistry which directly impinges on this work will be discussed in more detail in the following sections. Working with Ca 2+ as a messenger, molecules with a tendency to complex metal ions, called ionophores and possesing multiple ion dipole interactions to form a supramolecular interactions between ion and ligand will be regarded more closely.

Cation binding motifs.

One of the first supramolecular binding motifs for ions was accidently discovered by Pedersen in 1967. 20,21 Due to a catechol impurity in his starting material, he obtained as a side product, during the synthesis of a linear diol which he hoped would act as a ligand for the vandyl-ion, the so called dibenzo [18]crown-6 (2), which binds the potassium cation in its inner cavity. The synthesis of the first crown ether is shown in scheme 01. This gave rise to a new class of molecules which was named crown-ethers because of the crown-like shape.

Scheme 01: Serendipitous synthesis of the first crown-ether

The structural similarity to the biochemical valinomycin is evident and they have been used to bind a large variety of different cations throughout the years. Due to the spherical shape of inorganic cations, most cation-binding ionophores are of a macrocyclic nature. Selectivity for a specific ion depends on various factors. Some of these factors are the size complementarity between the cation and the host cavity which is depicted in figure 04. Furthermore, the electronic complementarity (HSAB theory), electrostatic charge, solvent polarity, degree of host preorganisation and the nature of the counter ion amongst others. 22 In nearly all cases cation binding is established through the interaction of the ion with Lewis-acids like ether oxygen or carboxylic acid oxygen. By altering the Lewis acid a different selectivity can be obtained. Ionophores bearing the soft sulphur atoms or nitrogen in their molecular backbone, tend to bind transition metals more strongly in many cases with respect to their oxygen counterparts. 23 Figure 04 : The influence of macrocycle size and the effect of the Lewis-acid nature on guest selectivity. From left to right, [15]crown-5 (3), [18]crown-6 (4) and [21]crown-7 (5) with their preferred guest ions. Copper (6) and silver (7) binding ligands with nitrogen and sulphur in their binding site.

The ion of interest for our study is however Ca 2+ . One of the first binding units which was used in the preparation of Ca 2+ -sensitive electrodes were phosphate esters. First attempts to prepare ionophores based on macrocycles were based on cryptands developed by Lehn and co-workers. The first cyclic ionophores were however not very selective. 24 A Ca 2+ -selective probe which was developed for the use in ion selective electrodes was ETH-1001 which resembles in terms of structure ½ of a crown ether and forms readily 1:2 and 1:3 complexes. 25 One of the most widely used chelating agents is ethylenediaminetetraacetic acid (EDTA, 8). It displays high affinity constants for nearly all divalent ions and its stong chelating properties are widely used in medicine during the treatment of metal ion poisoning. 26 The discrimination between Mg 2+ and Ca 2+ is most important in biological systems because the intracellular Mg 2+ concentration is about 4 -5 orders of magnitude higher than that of Ca 2+ . Therefore, ethylene glycol tetraacetic acid (EGTA, 9) was developed based on the design of EDTA (8) (see figure 05). EGTA (9) displays a large range of metal ion binding, like its predecessor but has a calcium binding constant which is about 5 orders of magnitude higher than the binding constant for Mg 2+ and is therefore very selective regarding these two ions. Some binding constants for EDTA (8) and EGTA (9) are depicted in table 03.

Table 03: Ion binding for EDTA (8) and EGTA (9), 25 °C, 0.1 M ionic strength (fully deprotonated)

Since biological systems are highly controlled environments and the presence of other divalent cations is rather low in the cytosol, EGTA (9) and its derivative 1,2-bis(oaminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA, 10) (see scheme 02) are the most widely use calcium chelators / ionophores up to date. The low binding constant towards magnesium is based on the small ionic radius (0.09 nm) of Mg 2+ which is the same size as Li + . Therefore the hydration state is much higher compared to Ca 2+ and ligand exchange occurs only slowly. EGTA (9) has been applied to prepare Ca 2+ -buffer solutions 27 and was applied in several biological tests, in order to study for example the involvement of Ca 2+ in brain ageing 28 or the control of various enzymes. 29 The BAPTA (10) molecule was first synthesized by Tsien and co-workers in 1980 and was developed as a pH insensitive alternative for EGTA (9) while maintaining the high selectivity for Ca 2+ over Mg 2+ . 30 The aliphatic amines of EGTA (9) which display a pK ass between 8.5 and 9.5, were replaced by aromatic amines with a pK ass of 5.97 and 6.36. This lower pK ass results in a larger proportion of non-protonated BAPTA (10) receptor under physiological conditions. Therefore, the Ca 2+ binding occurs faster since the EGTA (9) protons must dissociate first before Ca 2+ can bind, while BAPTA skips this step. EGTA (9) and BAPTA (10) are high affinity Ca 2+ binders with dissociation constants (K d ) between 50 nM to 100 nM. 31 Figure 05: Molecular structure of EDTA (8), EGTA (9) and BAPTA (10) Metal ion pK ass EDTA pK ass EGTA Mg 2+ 8.64 5.3

Ca 2+

11.0 10.9

Zn 2+ 16. 4 12.6

Cd 2+ 16. 4 16.5 After having defined the binding units of choice for Ca 2+ , the state of the art of triggered ion movement will be discussed.

Stimulus induced ion movement and release

In order to study ion transfer, an ion must be ejected or moved from its binding cavity.

Several different triggers to achieve ion movement are possible and the ones which were extensively studied are for example, a change in pH, binding of another ion or electro-and photochemical stimuli. The group of Shinkai showed that it is possible to mimic a syringe on a molecular level (11), see figure 05, where a silver ion is moved from one binding site, consisting of a crown ether, to another bearing a bis(ethoxyethoxy) unit linked by a 1,3alternate-calix [4]arene, upon increasing the pH of the solution. 32 Upon increasing the pH the amine in the more complexing crown ether part gets protonated leading to a decrease in the binding affinity towards the silver ion. The ion is then transferred through the alternate calix [4]arene tube as judged by dynamic NMR measurements, towards the bis(ethoxyethoxy) subunit which has after protonation a higher K a with respect to the protonated crown ether.

This pH-induced ion movement could be reversed upon addition of a base. Another example of a pH-triggered Cu 2+ -transfer (figure 06, 12) is the polyaza-macrocycle developed by Fabbrizzi and co-workers 33 . The Cu 2+ -ion, which is located at the pyridine-amine binding site at a pH between 4 and 5, can be transferred to the amide-amine binding site of the molecule by increasing the pH above 10.5. The pH induced deprotonation of the amides increases the affinity towards the ion and along with the translocation of the ions the macrocycle undergoes rearrangement to fit its cavity to the ion. A prominent way to trigger ion transfer in biological systems is the binding to another ion which leads to a rearrangement in the binding site or a change in the electron density and therefore releases or translocates the ion of interest. This chemically-induced ion translocation has the disadvantage that in order to move the ion acid or base needs to be added and side products can accumulate generally sitting in the amide-amine binding site of the molecule.

In order to achieve ion-triggered ion release one needs to look at the so called heterotropic negatively collaborative binding molecules. Rodriguez and co-workers synthesized a dicrown ether which is connected by a 2,2' bipyrazole linker (13, see figure 07). 34 In its initial state the molecule binds to a protonated diaminopentane. Upon the addition of a transition metal like Zn 2+ , the bipyridine linker undergoes a conformational change, forcing the crown ether units apart and liberation of the diamine (figure 07, left). Another example where a conformational change leads to a decrease in binding affinity is the dicrown ether (14) prepared by Costero and co-workers (figure 07, right). 35 In this molecule a smaller crown ether with 2 phenolic oxygen and a larger ring are connected via a dibenzolinker. The binding constant to sodium ions decreases in the presence of Hg(SCN) 2 which binds to the larger cavity, leaving the smaller cavity free. Due to the steric hindrance after binding to mercury only three of the five crown ether oxygens are free to bind to sodium leading to a decreased binding affinity.

Light and electrons offer two attractive means to trigger ion release mainly because they avoid the "waste build-up" in reversible systems. In addition, light can be applied cleanly and remotely with high temporal and spatial resolution. While electrons are interesting in redoxactive transition metal-based systems, they require wiring and electrode deposition during usage. Reversible photo-guided ion release is based on the combination of ionophores with photoactive groups, which change either conformation or their electron density upon irradiation. But before the photocontrolled release of ion will be discussed, a short introduction into photochemistry will be given to highlight the underlying principles.

1.5 A short introduction to photochemistry

Absorption of light

Photochemistry studies the evolution of an excited molecule after the absorption of a photon and the subsequent transformation, be it a reaction induced by the absorption of a photon or the emission of another photon of a different wavelength like fluorescence or phosphorescence. 36 The minimum energy required for the electronic excitation of a molecule is around 150 to 170 kJ/mol and corresponds to a red light with a wavelength of around 700 to 800 nm. Irradiation with lower intensities is in general not sufficiently energetic to provoke electronic excitation. The maximum energy for the electronic excitation is in an order of 590 kJ/mol and corresponds to ultraviolet (UV) light with a wavelength of around 200 nm.

Irradiation energies superior to that bear enough energy for bond dissociation.

Electronic transitions and excited state

A electronically excited state results from the excitation of an electron from the highest occupied molecular orbital (HOMO) to the lowest unoccupied orbital (LUMO) or a superior molecular orbital. A molecular orbital can be formed by linear combination of two s, s and p or two p atomic orbitals when they have a collinear axis of symmetry. Those bonds are called δ orbitals. A π molecular orbital is formed from p atomic orbitals overlapping laterally, while non-binding electrons are in the n orbital. 37 Transitions upon excitation with photons can occur from all of these ground state orbitals to the antibinding orbitals denoted with δ* or π*.

The required energy for these transitions increase in the following order:

n π* < π π* < n δ* < δ π* < δ δ*
In figure 08 a schematic representation of the excitation of an electron from the ground state to a singlet excited state through a n π* transition is given. When one of the electrons with opposite spin is excited from the ground state (S 0 ) in a higher orbital and the spin remains unchanged the excited state is called a singlet excited state (S n ).

In principle all excitation process are in first instance S 0 S n transitions meaning the electron spin is preserved. The excited electron can also undergo a spin change which leads to a so called triplet state (T n ) which is, according to Hund's rule, lower in energy (figure 09) . According to the Franck-Condon principle 38,39 the time required for an absorption of one quantum of light and for the transition of an electron into the excited state is as short as 10 - 15 s. Compared to the vibrational period of an atomic nucleus (10 -13 s) this time is rather short which means that an electronic transition occurs preferably to a vibrational state which has a wavefunction most similar to the state where it was excited from. This makes some transitions more likely than others and is responsible for the bands with different intensity in the absorption spectrum of a molecule. Fluorescence emission typically occurs from a singlet excited state (S 1 ) with a short lifetime of 10 -9 to 10 -7 s and is independent of the excitation wavelength. The displacement to longer wavelength of the fluorescence emission with respect to the absorption spectrum due to the loss of energy in the excited-state via vibrational relaxation is called a Stokes shift. 41 Internal conversion (IC) describes a radiationless deexcitation process on a time scale of 10 -11 to 10 -9 s and occurs mostly between different excited states like S 2 S 1 while internal conversion to the ground state can occur in molecules with low energy excited-states. Intersystem crossing (ISC) occurs on a time scale of 10 -10 to 10 -8 s and takes place between an singlet excited state and a close-lying triplet state. Intersystem crossing is therefore an internal change of the system including a electron spin change. Radiative deexcitation from the triplet excited state to the ground state is called phosphorescence and has a lifetime of 10 -6 to 1 s. The described processes are the principal deexcitation processes but several other deexcitation processes are possible like the transfer of an electron from another molecular fragment (PET) which will be described later on or a chemical conversion. The deexcitation process most pertinent for the presented work is fluorescence. The trans-isomer is stable and the cis-isomer is metastable meaning it thermically converts back to its initial state over time. The energy difference between the two state is about 50 kJ/mol. The distance between carbon 4 and 4' is 9 Å in the trans-isomer and 5 Å in the cisisomer. 43 This effect has been used for the synthesis of supramolecular structures such as dendrimers 44 or catenans. 45 Another example are diarylethene compounds. A representative which is of this class of photochromes ( 16) is shown in figure 12. 46 The open ring isomer is not planar since free rotation of the aryl-group (here thienyl, 16) is possible. The aromatic electrons are localised in the two thiophene units. 47 In the closed form the phtochrome (16-closed) possesses a cyclohexadiene moiety and the πelectrons are conjugated over the whole structure. 48

Light induced ion release

Some examples of ion-decaging ionophores harnessing photochromes will be presented after the introduction of a photo irreversible dacaging molecule. An example for irreversible phototriggered ion release was presented by Chen. 49 A phenolic Schiff-base derivative shown in figure 13 binds to Zn 2+ . Upon irradiation in the UV, the phenolic Schiff-base ( 17) can undergo a cyclisation reaction forming a 2-arylbenzoxazole. It was found that the binding affinity towards Zn 2+ is greatly reduced after the formation of the 2-arylbenzoxazole. As mentioned earlier, upon irradiation in the UV range azobenzene (15) undergoes a conformational change from its thermodynamically more stable trans-form to the cis-form.

The group of Shinkai implemented azobenzene into crown ether rings of different size (ranging from O 7 to O 9 ). 50 The cis-isomer of 18, which is depicted in figure 14a shows a relatively high binding constant towards alkali metals. Once it is irradiated with visible light it isomerises back to its trans-form and releases the bound ion. While the cis-isomer forms the typical crown-ether like loop and shows the appropriate binding affinity of crown ethers, the trans-isomer displays a largely decreased binding affinity with respect to all alkali metal ions.

The change in binding affinity is related to the linearly extended shape of the trans-isomer. A photoreversible proton ejector (figure 14b, 19) has been designed in the group of Jullien. 51 The azobenzene moiety consists of one ring with an acidic hydroxyl group induced by the presence of three chlorine atoms on the same ring and a second ring bearing a sulfonic acid in order to improve the water solubility of the molecule. The acidic hydroxyl proton is complexed with one of the nitrogens in the trans-form of the azobenzene. Upon irradiation in the UV the azobenzene undergoes isomerisation and hydrogen bonding becomes sterically unfavourable, thus releasing a proton The proton ejector (19) has been used to photocontrol the pH of an aqueous solution.

An example where a change in electron density, rather than a conformational change, is the trigger for ion release was given by Lapouyade and co-workers. 52 In this case an azacrown ether was used as the ion recognition unit which was connected to a non-symmetric dithienylethene as shown in figure 15. and therefore modulates the binding constant. This approach has different advantages compared to the azobenzenophane since both forms, open and closed, are stable at room temperature when not irradiated. However, both systems seem to work only in organic solvents and in order to mimic biological processes one would want to work under at pseudophysiological conditions. Another means to liberate ions upon irradiation are decaging or uncaging molecules which have been used to liberate ions such as Zn 2+ , Ag + 53-55 ,Cu 2+ 56 and biomolecules like glutamate 57 or cAMP 58 among others. A more detailed introduction to these types of molecules will be given in chapter 3.

Before ion sensors will be discussed, an introduction in the mechanisms of ion sensing by photoactive molecules utilising photoinduced electron transfer (PET) and photoinduced charge transfer (PCT) will be given in the next section.

1.7 Cation sensors and molecular sensing processes or clinical biochemistry 59 due to their involvement in numerous biological processes as highlighted earlier. This vast field of research has been reviewed in several articles. 59,60 Optical methods to detect ions has several advantages over other techniques due to being noninvasive relatively high increase of the output signal upon ion binding and the possibility to study the output with common microscopy and fluorescence techniques. Electrochemical methods for example have difficulties when it comes to small population of cells or single cell compartments. Therefore, the spatial resolution of fluorescence imaging methods make these type of chemosensors especially attractive.

concentration is based on the design of fluorionophores combining a fluorescent unit in one molecul the ionophore is an electron donor and the fluorophore is the electron acceptor. Upon cation binding the energy of the HOMO of the ionophore is decreased due to the presence of a cation and is now lower in energy than the HOMO of the fluorophore.

Fluorescence quenching is therefore suppressed and the singlet excited state can follow deexcitation via fluorescence emission. This process is called reductive PET.

Conversely, the principle of oxidative PET is shown in figure 17. Here, in the absence of a cation the electron in the LUMO in the excited state of a fluorophore can be transferred to the LUMO of the ionophore due to its appropriate energy level, which causes quenching of the fluorescence. Once bound to the ion the LUMO of the ionophore incre and is no longer available for the deexcitation process. Thus, the fluorophore can undergo deexcitation via fluorescence emission.

22

type of chemosensors especially attractive. [START_REF] Carafoli | Calcium as a Cellular Regulator[END_REF] The ability to detect a change in ion concentration is based on the design of fluorionophores combining an fluorescent unit in one molecule. In figure 16 an example of a fluoroinophore is given where the ionophore is an electron donor and the fluorophore is the electron acceptor.

Principles of photoinduced electron transfer (PET) of an off-on fluoroionophore by reductive PET.

the fluorophore does undergo a transition to the excited depicted using Frontier Molecular Orbitals at the top of figure 16. The singlet state of the fluorophore with an electron in the highest occupied molecular orbital (HOMO) and an ron in the lowest unoccupied molecular orbital (LUMO) could in principle emit fluorescence. In the shown fluoroinophore system, however, an electron from the HOMO of the ionophore can now be transferred to the HOMO of the fluorophore via PET causing scence quenching of the latter which is favourable due to the energy level of the orbital.

Upon cation binding the energy of the HOMO of the ionophore is decreased due to the presence of a cation and is now lower in energy than the HOMO of the fluorophore.

Fluorescence quenching is therefore suppressed and the singlet excited state can follow deexcitation via fluorescence emission. This process is called reductive PET.

Conversely, the principle of oxidative PET is shown in figure 17. Here, in the absence f a cation the electron in the LUMO in the excited state of a fluorophore can be transferred to the LUMO of the ionophore due to its appropriate energy level, which causes quenching of the fluorescence. Once bound to the ion the LUMO of the ionophore increases its energy level and is no longer available for the deexcitation process. Thus, the fluorophore can undergo deexcitation via fluorescence emission.

The ability to detect a change in ion ion binding and a fluoroinophore is given where the ionophore is an electron donor and the fluorophore is the electron acceptor.

on fluoroionophore by reductive PET.

the fluorophore does undergo a transition to the excited-state which is depicted using Frontier Molecular Orbitals at the top of figure 16. The singlet state of the fluorophore with an electron in the highest occupied molecular orbital (HOMO) and an ron in the lowest unoccupied molecular orbital (LUMO) could in principle emit fluorescence. In the shown fluoroinophore system, however, an electron from the HOMO of the ionophore can now be transferred to the HOMO of the fluorophore via PET causing scence quenching of the latter which is favourable due to the energy level of the orbital.

Upon cation binding the energy of the HOMO of the ionophore is decreased due to the presence of a cation and is now lower in energy than the HOMO of the fluorophore.

Fluorescence quenching is therefore suppressed and the singlet excited state can follow deexcitation via fluorescence emission. This process is called reductive PET.

Conversely, the principle of oxidative PET is shown in figure 17. Here, in the absence f a cation the electron in the LUMO in the excited state of a fluorophore can be transferred to the LUMO of the ionophore due to its appropriate energy level, which causes quenching of ases its energy level and is no longer available for the deexcitation process. Thus, the fluorophore can undergo For the study that will be described in this thesis Ca 2+ On the left side a "on-off" proton s developed in the group of de Silva is shown.

The fluorescence enhancement upon Ca 2+ addition was determined to be 30 fluo-1 (28-1). The inverse case was seen for the rhodamine chemosensor which showed an increase in fluorescence intensity of 15-fold for

). Ultimately those PET sensors gave rise to several similar fluoroionophores with substitutions on the fluorophore. Other fluorophores, such as or naphtalimide 77 harnessing PET to detect micromolar Ca have been developed over the years. Many BAPTA chemosensors are also commercially available and can be purchased in small quantities. Generally these PET sensors are used to understand biological processes involving Ca 2+ for example in growth factors.

Previous examples included only the case where fluorescence is increased upon metal ion binding but also the inverse case of PET systems are possible, the so called on which are much less popular than the previous case.

Photoinduced electron transfer principle of an "on-off" fluoroionophore A schematic representation of this process is given in figure 22. The photoinduced electron transfer in this case is more complicated but in general it can be said that in the absence of an ion the fluorescence is unquenched in the fluoroionophore and upon ion binding the orbital of the ionophore reaches a favourable energy level for electron transfer from the fluorophore to the ionophore which leads to fluorescence quenching. Two examples are shown in figure 23.

off" proton sensor (29) based on triphenyl substituted pyrazoline developed in the group of de Silva is shown. 79 be 30-fold for fluo-2

). The inverse case was seen for the rhodamine fold for rhod-1 (27-1) ve rise to several similar fluoroionophores with substitutions on the fluorophore. Other fluorophores, such as harnessing PET to detect micromolar Ca 2+ levels rs are also commercially available and can be purchased in small quantities. Generally these PET sensors are used to for example in growth factors. 78 ce is increased upon metal ion binding but also the inverse case of PET systems are possible, the so called on-off switches A schematic representation of this process is given in figure 22. The photoinduced electron transfer in this case is more complicated but in general it can be said that in the absence of an and upon ion binding the orbital of the ionophore reaches a favourable energy level for electron transfer from the fluorophore to the ionophore which leads to fluorescence quenching. Two examples are shown in figure 23.

) based on triphenyl substituted pyrazoline Another example of Metivier et al. 80 is depicted on the right of figure 23 and shows a dansylsubstituted calix-4-arene (30), the binding of this compound being found to be highly specific towards Hg 2+ showing PET from the dansyl fragments towards the ion centre when bound. No selectivity for other interfering cations like Na + , K + , Ca 2+ , Cu 2+ , Zn 2+ , Cd 2+ and Pb 2+ has been observed.

A generalized theory of electron transfer in chemical systems was introduced by Marcus 81 for which he received the Nobel prize in 1993. Concerning bimolecular electron transfer, after the excitation of a fluorophore it diffuses to the acceptor and forms an activated species. If electron transfer occurs between a fluorophore and an ionophore, a pair of radicals is formed, namely a radical cation and a radical anion. The electron transfer is then governed by kinetic and thermodynamic parameters. The Rehm-Weller equation (equation 04) 82,83 allows the estimation of the feasibility of electron transfer through readily quantifiable parameters.

Electron transfer only occurs if the Gibbs energy (∆G 0 ) for the reaction is negative or equal to zero.

ܩ∆ = ܧ- ௌ.௨. -ܧ ௗ.௨. + ܧ ை௫. -ܧ equation 04
Here, E S.Fluo. stands for the energy of the emissive excited state of the fluorophore, E red.fluo. is the reduction potential of the fluorophore, E Ox.iono is the the oxidation potential of the ionophore and E ion.pair is the energy of the coulombic interaction of the radical ion pair that is formed. Electron transfer reactions are generally very fast with respect to the lifetime of the excited state. The presented PET systems are an exception to the described bimolecular calculations since electron donor and acceptor are present in the same molecule. According to Marcus 81 the energy potentials of the donor state where the donor transferred parabolas (figure 24). The energy transfer takes place when the system is at an intersecting point (a). Marcus theory allows the calculation of time constant of the electron transfer (k from the activation energy (∆G equation 05 and 06.

݇ ா் =

In these equations H el is the electronic coupling between is the reorganization energy of the molecules which depends on the state and state of the product, k the absolute temperature. With this equation the speed of the electron transfer can be predicted for many systems.

the energy potentials of the donor-acceptor complex (R) and the charge separated state where the donor transferred an electron to the acceptor (P) can be represented in energy parabolas (figure 24). The energy transfer takes place when the system is at an intersecting point (a). Marcus theory allows the calculation of time constant of the electron transfer (k the activation energy (∆G ≠ ) or indirectly from the total Gibbs free energy (∆G

29

Photoinduced charge transfer (PCT)

Fluorophores containing an electron-donating group conjugated with an electron withdrawing group can undergo intramolecular charge transfer from the donor to the acceptor which is exalted upon excitation with light. A schematic example by Lord et.al. [START_REF] Lord | Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications[END_REF] fluorophore bearing a dimethyl amine as electron donating group and a 2dihydrofuran group as electron withdrawing part is shown in figure 25.

Schematic representation of a fluorophore capable of undergoing photoinduced charge transfer.

The resulting Stokes shift then depends on the environment and / or the microenvironment of the fluorophore. Therefore in fluoroionophores, binding a cation which interacts with the can alter the internal charge transfer within the fluorophore and the lting photophysical properties. [START_REF] Valeur | Chemosensors of Ion and Molecule Recognition[END_REF][START_REF] Rettig | Topics in Fluorescence Spectroscopy[END_REF] The different effects of cation binding on a donor or an acceptor site are described in figure 26.

Spectral displacements of PCT sensors resulting from interaction of a bound cation with an electron withdrawing group.

donating group conjugated with an electron withdrawing group can undergo intramolecular charge transfer from the donor to the acceptor which is [START_REF] Lord | Markers, Dyes, Nanoparticles, and Molecular Probes for Biomedical Applications[END_REF] of an anthracene -dicyanomethylenedihydrofuran group as electron withdrawing part is shown in figure 25.

capable of undergoing photoinduced charge transfer.

The resulting Stokes shift then depends on the environment and / or the microenvironment of the fluorophore. Therefore in fluoroionophores, binding a cation which interacts with the can alter the internal charge transfer within the fluorophore and the

The different effects of cation binding on a donor or an sors resulting from interaction of a bound cation with an electron-When an electron-donating group such as an amine interacts with an cation the positive charge attracts the electrons from the amine group and thus reduces the charge transfer character. As a result, the absorption and emission spectra should experience a blue shift along with a decrease of the extinction coefficient due to the decrease in conjugation caused by the cation. The dipole moment is therefore concomitantly decreased. In the opposite case when a cation interacts with the electron-accepting group, the electron-withdrawing character of this group is enhanced and therefore the dipole moment experiences an increase.

Absorption and emission spectra are in principle red-shifted and an increase in the extinction coefficient is often observed. The extent of this effect depends on the cation charge and the type of ionophore. For fluorophores that have a higher dipole moment in the excited state than in the ground state, cation-donor interactions destabilize the excited state more and lead to a blue shift. Conversely, if the cation interacts with an acceptor the excited state is more stabilized and leads therefore to a red-shift of the emission.

Most of the examples in the literature belong to the case where the cation interacts with a donor group. [START_REF] Kasner | [END_REF]88 Three examples based on an azacrown ionophore are given in figure 27. The two presented PCT-based ion sensors 31 developed by Mateeva et.al. [START_REF] Mateeva | Inclusion Phenom[END_REF] connecting the azacrown to a benzothiazolylpenyl unit and 32 using a merocyanine fluorophore developed by and Bourson et al. [START_REF] Bourson | [END_REF] were tested for spectral changes upon Ca 2+ binding. Upon ion addition, the absorption spectra of both fluorophores displayed a relatively large hypsochromic shift of the absorption maximum (31 = 360 nm 318 nm; 32 = 464 nm 398 nm). The change in the emission maximum is, however, much smaller (31 = 420 nm 418 nm; 32 = 621 nm 608 nm). The explanation for this lies in the nature of the excited state. Due to the positive polarization of the amine binding the cation, upon excitation a disruption of the bond between the nitrogen donor and the cation is induced. Thus, fluorescence is emitted from a species which is no longer (or only weakly) bound to the cation and therefore the fluorescence emission maximum is only weakly influenced. This interpretation is supported by several studies about the photophysics of this kind of PCT ion sensor. 91,92 The third ion sensor 33 bears the ion-sensing group in the acceptor position of the molecule due to the electron rich character of the dimethylamine unit. Thus, upon Ca 2+ -binding 33 gives a bathochromic shift in the absorption spectrum. Considerations made for the excited state equally apply here.

The aforementioned examples carrying an azacrown ionophore function well for Ca 2+binding but are not highly specific. Many other ions such as Li + , Ba 2+ or K + bind with the same or increased selectivity to the ionophore. Ca 2+ -selective PCT ion sensors are based on the BAPTA chelating agent developed by Tsien. 30 PCT-sensors have a distinct advantage over PET sensors since they can be internally calibrated against common microenvironental variables by dual excitation wavelength monitoring and observing the ratio between the two emissions. A lot of BAPTA-based PET and PCT sensors are commercially available nowadays due to their application in determination of cytosolic Ca 2+ concentrations. 93 Most prominent examples of BAPTA PCT sensors are shown in figure 28. In these fluoroionophores the fluorophore is a donor-acceptor molecule with an amine as the electron donor which is at the same time part of the ionophore and participates in the binding of Ca 2+ . Upon Ca 2+ -complexation in aqueous media, the absorption spectrum is blue-shifted with almost no influence on the emission spectrum. This accounts for all the BAPTA PCT sensors in the fura (35) and still (36) series. The indo (34) series however displays a rather large shift in the emission spectrum as well, which is rather untypical as we learned earlier.

The absorption and emission maxima in presence and absence of Ca 2+ for the three described molecules is shown in table 04. The same interpretation forwarded for the crown ethers applies here as well, meaning that the electron density of the aniline conjugated with the electron-withdrawing group of the fluorophore is reduced in the excited state and might become positively polarized which causes disruption between the cation and nitrogen binding. The possibility of photoejection in these molecules has not been examined yet. A study of the excited state revealed that the binding affinity of fura-2 ( 35) is three orders of magnitudes lower in the excited state. 94 As for the exception indo-1 (34), the PCT may not be sufficient to cause bond breaking between nitrogen and Ca 2+ . This would explain the shorter wavelength of the fluorescence emission compared to the other PCT sensors, indicating a less polar charge-transfer state.

As a last example, two multiple addressable systems based on the BAPTA ionophore and a pH sensing unit are presented. In figure 29 a presentation of two multi-addressable systems is given. The multiple addressable system 37 95 and 38 96 described by McClenaghan and de Silva found applications as molecular logic gates. As for 37, which consists of the a BAPTA fragment linked to a conjugated quinoline, Ca 2+ addition leads to an hypsochromic shift of the absorption spectrum while the decrease of the pH lead to a bathochromic shift of the absorption spectrum. By monitoring the absorbance at a certain wavelength a two input logic gate was designed. 38 displays as well the BAPTA fragment on one side and a diethyl amine on the other side of an anthracene fluorophore. In principle this system is addressable using the same ion / proton. When the PET is blocked on either side with H + or Ca 2+ characteristic anthracene emission can be observed yielding an AND logic gate when fluorescence is considered as an output. This is an example of the exploitation of PET for non-biological applications since most studies concerning these BAPTA based ion sensors involve the detection of cellular Ca 2+ . 97,98 Even though the field fluorescent ion sensing is rather old, research is still ongoing. Besides the continuous unravelling of the involvement of Ca 2+ in biological processes, 99 there is an interest in further improving the existing systems in terms of accessibility and utility. More recent publication on the field of Ca 2+ sensing include the modification of binding affinities by altering the core structure, 100 novel fluorionophores with improved emission wavelengths 101 and Ca 2+ decaging agents with improved decaging properties 102 or alternative triggers. 103 A recently published example of a series of novel PET Ca 2+ -sensor was developed by Collot et.al. 101 The molecular structure of the fluorionophores is shown in figure 30. The unique feature in the molecular structure of these Ca 2+ -sensors (39) is the presence of an aliphatic chain bearing an aromatic azide linked to the ethylene glycol part of the molecule.

Like that the aromatic ring was free to tune the binding affinity of the BAPTA unit.

Incorporating electron withdrawing groups like fluorine (39-F) and chlorine (39-Cl) decreased the binding affinity towards Ca 2+ from 3 µM (39) over 6 µM (39-F) to 22 µM (39-Cl). Fluorescence enhancement upon ion binding was in the range of 50-fold for the halogen derivatives and 60-fold for the parent molecule (39). The aliphatic azide chain had been subjected to "click"-chemistry in order to couple the ion sensor to substrates. The excitation maximum at 586 nm and emission maximum 604 nm gives rise to applications in multicolour imaging. Recent advance in Ca 2+ decaging agents will be discussed in chapter 3.

1.8 Biomimetic nanodomains and self-assembled cell mimicry

In order to study ion transfer in biomimetic nanodomains one needs to look at the cell mimicking assemblies that are available. This has been a vast field of research involving biologists and chemists in equal measure. Biologists, for example, have favoured a top-down approach by knocking out genes of an existing bacterium to obtain a cell with a minimum amount of genes in order to load them with synthetically created DNA. 104 Chemists on the other hand have been working on the bottom-up approach. Ever since the discovery that phospholipids are the major ingredient of the cell wall 105,106 they have been extensively

studied for the mimicry of biomembranes. 107 Years later it was found that these phospholipids are not the only organic molecules which are able to self-assemble into vesicles containing a biomembrane mimicking bilayer. Synthetic amphiphiles have been found to form bilayered structures in aqueous media as well. 108 The contribution of polymer chemistry to the selfassembled vesicles field is more recent. In 1999 Discher et al. 109 found that block copolymers consisting of a hydrophilic and a hydrophobic polymers can self-assemble to form polymer bilayers. These types of vesicles were called polymersomes and have ever since been exploited for the preparation of cell mimicry applications such as synthetic red blood cell mimics 110 or where engineered to posses the adhesive properties of leucocytes. 111 The three building blocks for cellular bilayer mimicry will be introduced in more detail in chapter 4. For now, it should be noted that there are vast selection of amphiphiles and polymers available to achieve the goal of cell mimicry and the subsequent biomimetic ion transfer studies in confined compartments.

Conclusion

The vast field of supramolecular chemistry holds the potential to impact a large variety of applications. The presented ion binding motifs in combination with photoactive groups like fluorescent dyes or photoswichtable groups give the opportunity to study ion transfer, which can be measured by fluorescence spectroscopy and microscopy techniques. Supramolecular assemblies using lipids, amphiphiles and polymer deliver the building blocks for the creation of cell mimicry. By combining the aforementioned disciplines of chemistry, a novel approach to artificial cell mimicry can be envisaged.

F 5 -BODIPY for selective labeling, fluorescence studies and surface patterning

Introduction

The BoronDIPyrromethene (BODIPY) fluorophore was first synthesized in 1968 by German chemists, namely Alfred Treibs and Franz-Heinrich Kreuzer. 1 The unsubstituted BODIPY skeleton, which is responsible for the molecules name was however not synthesized until the year 2009 and different routes of the synthesis had been published by 3 different groups. [2][3][4] The structure of the core compound ( 40) is given in Figure 30. The fluorescent properties of BODIPYs are generally defined by high quantum yields (0.2 -1) and high extinction coefficients ranging from 30000 to 80000 cm -1 M -1 depending on the substitution pattern. 5 Fluorescent emission occurs with a characteristic small Stokes shift and the dye displays a photostability which is often above those of frequently used fluorescence standards.

There are two main routes for the synthesis of BODIPY dyes which can be found in different variations throughout the literature. For the synthesis of symmetrical molecules a 3 step synthesis consists of the condensation of two pyrroles with an aldehyde leading to a dipyromethane, which is subsequently oxidized to a dipyromethene and complexed with BF 3 as shown in scheme 02.

Scheme 02: Synthesis of symmetric substituted BODIPY dyes.

For non-symmetrical BODIPYs a different approach was used to synthesize the final compound. The synthesis starts by the condensation of a pyrrole with an acylium equivalent, for example an acid chloride, an anhydride or an orthoester, leading to an acylpyrrole. 6,7 This intermediate is usually not isolated due to side reactions with other pyrroles in solution under acidic conditions. In a subsequent step, the acylpyrolle is reacted with a second differently substituted pyrolle to give the dipyrromethene and then complexed with boron trifluoride to form the asymmetric BODIPY. The synthesis is outlined in scheme 03.

Scheme 03: Synthesis of an asymmetric BODIPY Because of their relatively high chemical stability BODIPYs can be post-modified in several different ways which make them available for applications in labeling of proteins 8,9 or tuning of the fluorescence properties.

Post-functionalization of the BODIPY core

There are numerous ways for post modification of the BODIPY chromophore, which are linked to the aromatic nature of the molecule and are exploited in the literature. In figure 32 the positions of modifications are highlighted. The α-position is easily modified by nucleophilic aromatic substitution reactions due to the electron-withdrawing effect of the partially positively charged nitrogen in proximity.

Monohalogenated derivatives had been synthesized by Dehaen and co-workers and were subsequently functionalized by different nucleophiles using Pd-catalyzed crosscoupling. 10 In this manner the fluorescent properties of the dye could be tuned with emission maxima ranging from 503 -578 nm. In the same manner the β-position, substituted by an iodine, has been functionalized with conjugated aromatic groups, to change the spectral properties.

Another example from the same group uses S N,Ar of a chlorine in the α-position of BODIPY These elongated BODIPYs show red shifts in absorption and emission spectra leading to near infrared emitting dyes. The influence of the solvent polarity is also increased suggesting a more polar excited state of these structures 12 .

Scheme 05: Knoevenagel type reaction on a BODIPY bearing methyl groups in the α-position.

The β-position is the most electron rich position of the fluorophore, which can be shown by the mesomeric structures. Therefore this position is susceptible to electrophilic aromatic substitution. The classical examples can be found in the literature namely sulfonation (48), nitration (49), formylation (50) and halogenations (51). While sulfonation is mostly applied to increase the water solubility of the dye while maintaining its spectral properties, 13 formylation 14,15 and halogenation 16 lead to intermediates which can be further modified.

Bromination of the BODIPY core showed a red-shift in absorption and emission spectra and fluorescent quantum yields were decreased due to the heavy atom effect. An overview of the S E,Ar reactions is given in figure 33 showing a schematic representation of standard BODIPY (47) in the center. (53) or disubstituted products (54) when reaction times are prolonged and excess nucleophile is used. 17 The reaction yield decreases when more sterically hindered alcohols are used. The second way to make the boron react is the activation of the boron center itself by strong Lewis acids like AlCl 3 leading to disubstituted reaction products (56). 18 Other examples include the use of organometallic compounds like organolithium or Grignard-type reactants. 19 Scheme 06: Substitution of fluorine at the boron center. left: basic conditions, right: acidic conditions.

In next few pages a new BODIPY based chromophore is introduced with a new way to post functionalize the BODIPY skeleton based on the chemistry of a pentafluoro phenyl ring.

Perfluorophenyl rings are known to undergo S N,Ar reactions due to the electron withdrawing effect of the fluorine atoms. This chemistry has been extensively exploited for different application for example in post-modification of polymers or the facile introduction of side chains onto a porphyrin ring system. Step one being purification by column chromatography with silica as the stationary phase and the second step being crystallization from chloroform. This proved to be necessary due to the instability of the title compound on the chromatography column. This instability can be explained by the unsubstituted β-position, which seems to be unstable under the slightly acidic conditions on the column. Derivatives with an ethyl group in this position were chemically more stable and are shown later on in this manuscript. A representation of the synthetic pathway is given in scheme 07. The overall reaction yield was 33 % with respect to the perfluorobenzaldehyde (57).

Scheme 07: One-pot synthesis of F 5 -BODIPY (59).

Single crystals suitable for X-ray diffraction were obtained from chloroform. The formation of single crystals of BODIPYs has recently been reported from acetonitrile. 22 The crystallographic structure of F 5 -BODIPY (59) shows a planar BODIPY moiety and the out of plane pentafluorinated phenyl ring. The phenyl ring is oriented in a 90.01° angle in the solid state with respect to the BODIPY plane, which is an important factor in assuring the high fluorescence of the molecule. The steric hindrance of the methyl groups along with the more bulky fluorine atoms compared to hydrogen atoms is the mainly the reason for the exact perpendicular orientation. On comparing with a related hydrocarbon analogue like a para bromophenyl fluorine atoms are replaced by hydrogen resulted in a decrease of the angle to 78.91° and the dimethylamino analogue showed an angle of 87.61°

S N Ar on the perfluorophenyl ring of F amine conjugates

As described in the introduction, perfluorinated phenyl rings a susceptible to nucleophilic aromatic substitution, 24 offering an effective reaction pathway to C formation. This offers an alternative to metal way of modification to the BODIPY core. Th able to undergo those kind of modifications. However, as presented in the introduction, relatively harsh conditions need to be applied to make the boron react and act the nucleophile or the boron centre itself is needed. S room temperature under mild conditions in DMF and advancement of the reaction was As a second example for a biologically interesting reactive functional group, a primary amine was chosen as a representative for amine bearing amino acids in proteins like lysine. As in the previous experiment, the reaction of 59 with an excess of butyl amine (8 fold) was monitored by means of HPLC. The reaction proved to be slower compared to the substitution with ethane thiol under basic conditions. Equally, no side products were observed. The retention time of the BuNH-BODIPY (61) was even longer (8.3 min) which is due to the free hydrogen in the secondary amine. Recording the 19 Upon heating to 120 °C overnight and using 40 eq. of diethyl amine, the substitution in the para-position could be forced towards the substitution product Et 2 N-BODIPY (62). Unlike the case with a primary amine, side products were observed and careful purification by column chromatography and crystallization was necessary to obtain the pure product. A direct comparison of the reaction progression of F 5 -BODIPY (59) with amines and thiol as judged by the integrated peak areas in the HPLC chromatograms is depicted in figure 36, highlighting the increased reactivity of 59 towards thiols. The reaction of 59 in presence of ethane thiol (5 eq.) and butylamine (8 eq.) gave quasi-exclusively the thiol product after only 5 min reaction time. This is due to the increased solubility of amine base in DMF compared to K 2 CO 3 and therefore more efficient deprotonation of the thiol as shown in figure 36 on the right side. The selectivity and high reactivity of thiols with the perfluorophenyl moiety under basic conditions has been demonstrated. The reaction of 59 with ethane thiol in DMF without adding a base is much more sluggish and gave a conversion of only 54 % after 5 days at room temperature. In a 1:1 (v/v) mixture of DMF in water conversion proved to be equally efficient regarding the selectivity of thiol over amine. In fact the amine conjugate seemed to be unreactive under these protic conditions. However the reaction proceeded much slower showing a total conversion of 89 % after 27 h. Reaction progression as judged by HPLC peak intensities is shown in figure 37. 1 % amine product (61) In order to improve the reaction rate, additional charged groups like sulfonic acids or carboxylic acids could be incorporated into the BODIPY-skeleton to improve the water solubility, which might ultimately lead to a faster conversion.

2.3 Spectroscopical studies of F 5 -BODIPY (59) and its substitution products (60,61 and 62) The spectral properties of parent BODIPY (59) and its substitution products (60,61 and 62) have been measured in air-equilibrated THF, a relatively polar solvent which efficiently solubilises all the chromophores. The parent molecule shows a high molar extinction coefficient as shown in figure 38. Emission following irradiation at 488 nm occurs with a small Stokes shift of 413 cm -1 , which is typical for BODIPY fluorophores (ca. 400 cm -1 ). The quantum yield was determined by comparison to a reference standard which was chosen to be fluorescein in 0.1 M NaOH (aq.) with a quantum yield of 0.95. 25 The fluorescein was chosen as reference as it offered a similar absorption and emission range to the molecules under investigation. The quantum yield of BODIPY (59) was determined to be 0.98, reaching almost unity. The absorption and emission spectra (figure 38) of the para-position substituted BODIPYs are almost unchanging with respect to the parent molecule showing similarly high molar extinction coefficients for the lowest energy band and fluorescence quantum yields (0.90 -0.96). For better comparison these results are listed in table 04. The absorption spectra of these fluorophores are almost coincident with widely used argon ion laser, which emits at 514 nm and remote substitution has only a negligible effect on the absorption spectra, making this method for ad hoc introduction of the fluorophore a viable alternative to known methods like ester formation where the emission spectra and quantum yield is altered from the acid. 26 Relatively few fluorophores are perfectly adapted for this wavelength and are often expensive like commercially-available "Alexa Fluor 514". Fluorecence lifetimes for molecule 59 -62 were obtained by time-correlated single photon counting spectrometry. An example of emission from F 5 -BODIPY ( 59) is given in figure 39 showing the photon counting on the left and the fitting of the lifetime and distribution of residuals and the standard deviation on the right. The strongly allowed nature of the S 0 S 1 transition, as denoted by the strong molar extinction coefficients allows estimation of the excited-state lifetime using a Strickler-Berg analysis as denoted in equation 07. In this equation n is the refractive index of the solvent (assumed to be the same in all spectral regions), <ν F -3 > -1 is the reciprocal of the mean value of Einstein law, each photon absorbed by a chemical system, only one molecule is activated for a following reaction. According to this law of photo equivalence equation 09 can be formulated and shows that the number of molecules excited (N the number of photons absorbed (K abs ). K is the incident photon number and can be derived The quantum yields for photodegradation (Q fluorescein isocyanate which is in the same order of magnitude as a literature value in deoxygenated EtOH. 30 The Q The quantum yields for photodegradation (Q photoproduct ) were determined to be 7.5 fluorescein isocyanate which is in the same order of magnitude as a literature value in

ܰ ௫ = ܰ ௫ ሺ1 -ܳሻ equation 11 ܰ ௫ -ܰ ௫ = -ܰ ௫ ܳ
The Q photoproduct for molecule 60 -62 were estimated to be 2.7 ) were determined to be 7.5×10 -5 for fluorescein isocyanate which is in the same order of magnitude as a literature value in were estimated to be 2.7×10 to non or low emissive states, such as triplet states, 31 photoisomers 32 or other metastable species (for example geminate radicals or species originating from proton or electron transfer). [33][34][35] For 50 % of 82 molecules of 1 the peak emission intensity reflecting the brightness was determined to be at least 5.3 x 10 3 ௧௦ ௦

and could reach values up to 2.9 x 10 4 ௧௦

௦

. These values are in good agreement with an upper limit of 2.9 x 10 5 ௧௦ ௦

36

.

The lower values measured arise from the random orientation of the molecules relative to the excitation beam polarization and the instrumental detection efficiency. From 50 % of the molecules at least 2.1 x 10 4 photons and in the best case up to 2.4 x 10 5 photons have been emitted before photobleaching (figure 44). Considering the limitations of system, as mentioned previously, the measured value of ଵ ொ ೠ = 3.7 x 10 4 photons in solution reflects the results obtained in the single molecule studies and is in good agreement with known organic emitters. 36 The emission occurs within 11 seconds for 50 % of the molecules and the duration can reach up to one minute (figure 43b). An exponential decrease in the duration of until photobleaching is observed from 0 to 45 seconds, while a second population of molecules is observed emitting photons longer (55 s -60 s). The distribution of the decay times of 59 is highlighted in figure 43c. The average lifetime deduced from this experiment is 5.1 ns with a standard deviation of 0.9 ns and a peak at 4.9 ns. This is in accordance with the measured lifetime value of 5.8 ns in bulk solution but somewhat slightly shorter. This could result from an enhanced non-radiative deactivation of the excited states in the polymer environment. As in figure 43b, a second population of single molecules displaying a lifetime of 6.8 ns is observable. By plotting the number of photons emitted over the fluorescence lifetime (figure 43d) it becomes evident that a small number of molecules possessing a longer fluorescence lifetime also emit a higher average number of photons. In a previous study of single molecules in PMMA 37 the same observation has been made and the minor population of molecules had been attributed to molecules embedded in the more rigid micro-environments of PMMA. BODIPY 59 shows therefore some sensitivity to the environment and can respond with a change in fluorescence lifetime and the average number of photons emitted while preserving suitable emissive properties.

Conclusion

Selective functionalization of a perfluorophenyl bearing BODIPY (59) has been shown and selective labeling with thiols over amines has been accomplished via C-N and C-S bond formation. These photostable conjugates show strong absorption and emission in organic solvents. In aqueous mixtures, high selectivity for reaction with thiols conspires with a 90fold higher quantum yield of the corresponding conjugate (EtS-BODIPY (60) cf. BuNH-BODIPY ( 61)), which makes the thiol conjugate especially attractive for the labeling of amino acids in proteins. Indeed, this corresponds to a circa 10 4 contrast on a -NH 2 versus -SH site basis. In principle, perfluorophenyl chemistry can be exploited for other fluorophores in the same fashion it has been presented here. Another application could be the labeling of surface using the perfluorphenyl BODIPY 59 to introduce in a simple fashion functional groups, which can be used for example in surface patterning applications which is presented in the following section.

Perspectives and preliminary results of photoinduced surface patterning using an azide-BODIPY (63) and click chemistry

Due to the high photostability and unique reactivity of the F 5 -BODIPY (59) several applications can be envisaged. Here some preliminary results obtained for perfluorophenylazide-BODIPY based on the molecular structure of F 5 -BODIPY (59) in the application of photoinduced surface patterning are presented. Several click and click-like reactions are known to date. Here a short introduction to photoinduced click-like reactions and application in surface patterning is given. For example, in the group of Barner-Kowollik 38 the photoreaction between a maleimide unit and a 2-formyl-3-methylphenoxy (FMP) group has been exploited to immobilize a bromide-bearing maleimide derivative on silicon wafers functionalized with FMP. A photomask was applied so that a pattern was produced on the surface upon irradiation of a FMP surface at 320 nm in a solution containing the brominated maleimide. The most prominent example for click reactions is the cycloaddition of an azide to an alkyne which was developed in the groups of Sharpless 39 and Meldal 40 . The approach of an photoinduced generation of an Cu I using a photoinitiator to generate the cycloadduct with spatial resolution has been shown in the group of Bowman. 41 A Cu II pre-catalyst in a solution containing polyethylene glycol (PEG)-alkyne was irradiated in the UV in the presence of a photoinitiator on the surface of an azide-functionalized polypropylene on a silicon substrate.

By using a photomask, spatial patterning of the cycloadduct was achieved. A schematic representation of the process is given in figure 45 Perfluorophenyl azides have also been used for the direct surface modification of polymer films. 42 Therefore the reactivity of the azide group, namely formation of a nitrene and incorporation into C-H bonds or addition to olefins upon irradiation was used to attach them covalently to the polymer film. A last example which excludes the use of light but achieves the patterning of a surface without the use of a mask is shown in the group of Stoddart. 43 An atomic force microscopy tip was coated with copper and alkynes in a solution on top of an azide-functionalized surface was patterned by writing the desired pattern via moving the AFM needle over the surface. The approach taken in this preliminary study is a combination of the photoinduced click reaction on surfaces and the "writing" of a pattern on a surface.

Chemistry of azide-BODIPY (63) and photoinduced click reaction in solution

The synthesis of the azide-BODIPY ( 63 The photoinduced click-reaction was tested using the photoreducible copper(II)-tren complex (64) developed in the group of Vincent and published in several papers by Harmand et.al. 44,45 The photolatent catalyst was kindly provided by the group and the molecular structure is shown in scheme 09. Upon irradiation in the UV the ketoprofenate provokes a monoelectronic oxidation of the ligand due to the quenching of the triplet state via charge transfer. The ligand abstracts then a proton from the solvent leading overall to an reduced metalcomplex, a protonated ligand and a hydroxyl radical. It has been shown that the catalyst can be activated by UV light and stopped by purging the reaction with oxygen leading to an oxidized metal center. The click-reaction could be restarted after depletion of oxygen upon irradiation in the UV. In a first test, the reaction between azide-BODIPY ( 63) and dodec-1-yne was tested in solution using [Cutren(CH 2 PhtBu) 3 ](kt) 2 (64) (see scheme 10). All reactants and the catalyst were dissolved in toluene, deoxygenated by argon bubbling and irradiated for 28.5 h at 365 nm using a classical TLC lamp. By that time the reaction had proceeded to 78 % completion, and was stopped and purified. The reaction product was obtained in 60 % yield after column chromatography. The reaction scheme leading to the click-product triazole-BODIPY ( 65) is shown in scheme 10. The reaction proceeded rather slow most likely due to the larger extinction coefficient of the BODPY fluorophore (52 000 cm -1 M -1 ) 46 and therefore much less incident light reaches the catalyst. In principle it has been shown that the cycloaddition between azide-BODIPY ( 63) and a terminal alkyne can be catalysed by [Cutren(CH 2 PhtBu) 3 ](kt) 2 (64). The reaction yield was sufficient for some proof-of-principle experiments to pattern the azide bearing BODIPY onto alkyne-functionalized surfaces. In order to verify that the click product is still fluorescent and at an observable wavelength range, electronic absorption and emission spectra of 65 were recorded in ethanol and the graphs are shown in figure 46. The absorption maximum can be found at 543 nm and the emission occurs with a typically small Stokes shift of BODIPY fluorophores at 561 nm. An extinction coefficient of 57 000 M - 1 cm -1 has been measured and is only a little lower than previously mentioned BODIPY dyes (59 -62).

2.5.2 Surface patterning using azide-BODIPY ( 63) and [Cutren(CH 2 PhtBu) 3 ](kt) 2 (64) A precise description of the confocal fluorescence microscope setup used for the experiment is given in the experimental section. Low density (LD, 10 9 -10 10 alkynes/cm 2 ) and high density (HD, 10 14 alkynes/cm 2 ) on polyethylene glycol coated glass surfaces were purchased from Micro Surface Inc. (http://www.proteinslides.com) and used as bought. The surface patterning process is described in figure 47. A solution containing 50 mM azide-BODIPY (63) and 1 mM [Cutren(CH 2 PhtBu) 3 ](kt) 2 (64) in toluene was placed on top of the alkyne-coated surface. Irradiation was performed from the bottom of the coated glass cover slide with a 375 nm laser source. After irradiation the solution containing the azide (63) and the catalyst (64) were removed and the coated glass cover slide was cleaned via 3 cycles of sonication in ethanol using a classical ultrasound bath. Subsequently, the process was repeated in toluene. The now BODIPY (63) -functionalized fluorescent glass surface was observed via confocal microscopy using the observation setup described in figure 48. The first observation is that the fresh LD resulting from impurities or defects on the surface, see figure 49 left. However, the LD surface that was subjected to irradiation shows a clear stepwise i higher fluorescence on the irradiated side and a side with lower fluorescence corresponding to the position of the photomask. It should be noted that a certain fluorescence intensity is also seen on the non-irradiated part of difference in fluorescence is nevertheless specific.

In following step a new LD-surface was used and covered with a toluene solution of azide (63) and catalyst (64). In figure 50 the conducted exper the obtained fluorescence lifetime imaging microscopy (FLIM) image is shown on the right.

A laser beam at 375 nm with a power of 400 "write" squares with a size of 20 x 20 scanning the same area several times with the same constant speed of 1.2 of a constant CW power of 400 corners correspond to scanner parking. The FLIM images on the right side of figure 50 show the fluorescence intensity in terms of brightness as well as the fluorescence lifetime of the emitted light. First it is seen that an increased the number of passages results in a brighter fluorescence emission with a maximum at 3 passages. An additional passage seems not to further increase the fluorescence emission even though that needs to be considered with care since some non-coplanarity of the substrate vs scanner leads to a loss in intensity as well. The fluorescence lifetime is in the range of 5 ns as expected of BODIPY fluorophores. At the point where the irradiation started a shorter lifetime of around 4 ns is observed with much higher intensity, which will be discussed later on.

A similar experiment was conducted using the high-density alkyne functionalized surface (HD-surface) mentioned above. Again 4 squares (20 µm x 20 µm) were patterned on the surface using a confocal laser beam at 375 nm. In addition to the 4 squares, a series of 8 points with increasing irradiation times per point were patterned with a laser power of 400 nW (left to right) and 10000 nW (top to bottom) as described in figure 51 Unlike in the previous experiment, the laser power was varied for the squares rather than the number of passages. 400 nW (top left), 1000 nW (top right), 5000 nW (bottom left) and 10000 nW (bottom right) were used to prepare the squares with a passage time of 1.2 ms/pixel at 375 nm. After the described washing procedure the surface was observed under the confocal microscope. In the fluorescence image it is visible that the increasing laser power yields an increase in fluorescence on the HD-surface. Laser powers between 5000 and 10000 squares with respect to the background. The square produced with a laser power of 1000

shows a brighter fluorescence at the edges of the square where the laser changed its direction and therefore stayed longer in place. The characteristic where the irradiation started is present on the HD 10000 a.u. even shows the shorting of the lifetime along the whole edge. A explanation could be a secondary photoreaction not surprising since the perfluorophenyl azide unit is photoactive on its own and forms reactive nitrenes upon irradiation.

with 400 and 10000 nW and increasing irradiation times is given in figure 52. where they reach saturation and no further increase is noted anymore.

Since the spatial resolution of the surface patterning should in (64) and azide, a solution of 50 (64) was prepared in cyclohexanol which is a rather viscous solvent (41.07 mPa.s). The experiment was then conducted using the previousl surface with the azide / catalyst mixture and preparing squares and points via irradiation at 375 nm in the confocal volume. The plan of the experiment is shown in figure 53 on the left.

67

In the fluorescence image it is visible that the increasing laser power yields an increase in surface. Laser powers between 5000 and 10000 nW yiel squares with respect to the background. The square produced with a laser power of 1000

shows a brighter fluorescence at the edges of the square where the laser changed its direction and therefore stayed longer in place. The characteristic shortening of the lifetime in the corner where the irradiation started is present on the HD-surfaces as well. The square produced with a.u. even shows the shorting of the lifetime along the whole edge. A explanation could be a secondary photoreaction which occurs in places with longer irradiation times which is not surprising since the perfluorophenyl azide unit is photoactive on its own and forms reactive nitrenes upon irradiation. 47 Analysis of the fluorescence intensity of the point series nW and increasing irradiation times is given in figure 52.

Left: Fluorescence intensities of points created with 400 nW laser power. Right: Fluorescence intensities of points created with 10000 nW laser power. Image obtained by scanning the surface with the observation setup shown in figure 48 with a speed of 0.600 ms/pixel.

For the points prepared with 400 nW, 375 nm irradiation the fluorescence intensity increases almost linearly with increasing irradiation times. No further signal increase is noted for the s. The dots prepared with 10000 nW. show an increase until 6

where they reach saturation and no further increase is noted anymore.

Since the spatial resolution of the surface patterning should involve the diffusion of catalyst

) and azide, a solution of 50 mM azide-BODIPY (63) and 1 mM [Cutren(CH ) was prepared in cyclohexanol which is a rather viscous solvent (41.07 mPa.s). The experiment was then conducted using the previously described procedure, covering the HD surface with the azide / catalyst mixture and preparing squares and points via irradiation at nm in the confocal volume. The plan of the experiment is shown in figure 53 on the left.

In the fluorescence image it is visible that the increasing laser power yields an increase in nW yield very bright squares with respect to the background. The square produced with a laser power of 1000 nW shows a brighter fluorescence at the edges of the square where the laser changed its direction shortening of the lifetime in the corner surfaces as well. The square produced with a.u. even shows the shorting of the lifetime along the whole edge. A explanation could which occurs in places with longer irradiation times which is not surprising since the perfluorophenyl azide unit is photoactive on its own and forms Analysis of the fluorescence intensity of the point series nW and increasing irradiation times is given in figure 52. ) was prepared in cyclohexanol which is a rather viscous solvent (41.07 mPa.s). The y described procedure, covering the HDsurface with the azide / catalyst mixture and preparing squares and points via irradiation at nm in the confocal volume. The plan of the experiment is shown in figure 53 on the left.

The laser power was varied for the 4 squares using 400 nW (top left), 1000 nW (top right), 5000 nW (bottom left) and 10000 nW (bottom right). All squares were prepared by scanning the laser at 375 nm over the square area with a speed of 1.2 ms/pixel. In between the squares several points have been patterned using 400 nW (top line from left to right) increasing the irradiation time from 1 to 13 s, 1000 nW (bottom from left to right) increasing the irradiation time from 1 to 13 s and 5000 nW (top to bottom) increasing the irradiation time from 1 to 12 s. The obtained image after the washing procedure is shown in the middle for figure 53.

The obtained image was, despite the higher viscosity of the solvent, unexpectedly rather blurry. The square obtained with 400 nW is not visible in this experiment. Between the square obtained at 1000 nW and 5000 nW an increase in fluorescence intensity is noted along with a shorter lifetime at the corner of the square. The square obtained with 10000 nW irradiation shows a shortened lifetime for the whole square area and a reduced fluorescence intensity. The points in this experiment are not sufficiently well-resolved to allow a single analysis by intensity profiles. The points obtained from top to bottom using a laser power of 5000 nW show a shortening of the lifetime between approximately 3 and 4 s irradiation time. The fluorescence emission spectra of the square obtained with 1000 and 5000 nW show the unaltered emission spectra of the BODIPY (65). The emission spectra of the square obtained with 10000 nW irradiation, where the lifetime is tremendously shortened also shows a different emission spectra with a non-negligible red shift, which supports the hypothesis that the shortening of lifetime correlates to the formation of a photoproduct or the growth of a second band. Overall it can be said that the resolution of the pattern become worse in cyclohexanol and the fluorescence intensity decreases with increase in irradiation time, which is the opposite effect as observed in toluene.

Summary

In summary, an application for the perfluorinated BODIPY fluorophore has been shown

harnessing the facile introduction of an azide group in para-position of the perfluorophenyl ring. Proof-of-principle results have been obtained for surface patterning by means of photolithography using this azido-BODIPY dye (63) in concert with a photoactivatable Cu(II)precatalyst (64). The straightforward micrometre-resolution patterning on the surface using confocal laser excitation without recourse to a photomask shows a distinct advantage over many known systems.

The system depends on many parameters. Increasing viscosity seems to decrease the resolution while the increase of the excitation power leads to more intense labeling in toluene and to photobleaching in cyclohexanol. Longer irradiation times lead to more patterning in toluene but to photobleaching in cyclohexanol, leading to the assumption that the diffusion parameter is indeed not yet understood and further studies need to be conducted. The presence of oxygen is another factor which seems to inhibit the patterning process completely (experiment not shown).

Synthesis and characterisation of fluorescent Ca 2+sensors and photolabile Ca 2+ -decaging agents

As previously outined, one final goal of the project is the demonstration of ion transfer between nano-objects in confined compartment to mimic rudimentary cellular communication. In the general introduction some fluorescent ion sensors and photolabile ion ejectors were described. In chapter 1 the properties of BODIPY fluorophores were discussed and a novel fluorophore based on a perfluorophenyl moiety bearing BODIPY has been introduced. In this chapter the focus lies on the design of a fluorescent ion switch based on a BODIPY fluorophore, harnessing the unique spectral properties of this molecular unit and combining it with an ion-sensor. Equally, the design of amphiphilic photo-decaging molecules is presented using a Ca 2+ -chelator and subsequently, on combining these fuctional molecules ion transfer is established under pseudo physiological conditions with micromolar concentrations of calcium.

3.1 Synthesis and spectral studies of water soluble Ca 2+ -sensors based on BAPTA and BODIPY

Synthesis of BAPTA-BODIPYs (72 and 76)

The BAPTA binding unit was chosen for its high affinity constant and selectivity towards Ca 2+ over Mg 2+ under physiological conditions of ionic strength and pH. In this receptor, which comprises aniline units rather than the aliphatic amines in EGTA, shows less pHsensitivity in the physiological pH range (6.5 -7.5) due to the conjugation of the aniline electron pair with the benzene ring. Equally this unit, with an accessible oxidation potential can be exploited for photoinduced electron transfer processes, for example as a means to switch the fluorescence of an adjacent fluorophores via reductive quenching offering the means to develop selective fluoroionophores. The multi-step synthesis of the BAPTA receptor, whch would subsquently serve as a synthon in the elaboration of more functional molecules, was carried out according to a published procedure by the group of Tsien. 1 The synthesis is presented in scheme 11

Scheme 11: Synthesis of methyl-BAPTA precursor (69).

The potassium salt of o-nitrophenol (65) was formed in methanol using potassium hydroxide and subsequently reacted with a large excess (10 eq) of 1,2-dibromoethane to obtain the mono-substituted bromoethane (66) with a yield of 37 %. Generally, the yield for this reaction rarely surpasses 45 % due to the formation of disubstituted product even while working with a high excess of reactants and upon slow addition of 65 as well as a possible elimination reaction. In order to obtain a BAPTA derivative which is susceptible to reactions only on one of the two aromatic rings, 66 was reacted with an o-nitrophenol carrying a methyl group in the para-position with respect to the nitro-group. Thus the potassium salt of 5-methyl-2nitrophenol was formed with KOH in MeOH and was then subsequently reacted with intermediate 66 to form the dinitro intermidaite (67) with a yield of 67 %. A Pd/C catalysed hydrogenation led to the diamine intermediate (68) with a yield of 71 % and subsequent alkylation of the amines, using ethyl bromoacetate under basic conditions in acetonitrile with sodium iodide as catalyst, formed the methyl-BAPTA precursor (69) (35 % yield). The unoptimized reaction yield was rather low due to the short reaction time which was used during the procedure which could otherwise lead to more product, for example on adding periodically multiple aliquots of the ethyl bromoacetate.

Starting from methyl-BAPTA (69), the synthesis of two different water soluble BAPTA-BODIPY fluorescent ion sensors was envisaged. The synthesis is outlined in scheme 12 for BAPTA-BODIPY (72) and scheme 13 for BAPTA-PH-BODIPY (76) bearing a phenyl linker between ionophore and fluorophore. The phenyl spacer is anticipated to better separate the discrete molecular subunits thus affording a greater degree of predictability in the behaviour of complexation and switching, while the molecule where the molecular subunits are directly connected case could give a more dramatic switching performance.

N O O N 69 O O O O O O O O N O O N 70 O O O O O O O O O H POCl 3, DMF 74 % N O O N 71 O O O O O O O O N N F B F N O O N 72 O O O O O O O O N N F B F LiOH quantitative o-chloranil H N BF 3 -Et 2 O 26 %
Scheme 12: Synthesis of BAPTA-BODIPY (72) Methyl-BAPTA (69) was formylated using a Vilsmeier-Haack procedure 1 using POCl 3 and DMF. Following a mild hydrolysis, the resulting aldehyde ( 70) could be obtained in a good isolated yield (74 %). The ester protected BAPTA-BODIPY ( 71) was obtained following a known three step, one-pot procedure. For BAPTA-PH-BODIPY (76) a different synthetic approach was adopted. The synthesis started from the methyl-BAPTA precursor (69) and the reactive aromatic ring was brominated in the para position of the aniline using Br 2 . 4 The brominated BAPTA intermediate ( 73) was obtained with a yield of 70 % after purification. Reaction at the bromine-bearing carbon of 73 allowed conversion into a boronic ester following the Miyaura methology. 5 The BAPTA boronic ester intermediate (74) was obtained reacting the bromine (73) with bis(pinacolato)diboron under microwave conditions employing a PdCl 2 (dppf) ([1,1bis(diphenyphosphino)ferrocene]-dichloro palladium) catalyst and potassium acetate as base with a yield of 65 %. Indeed, this intermediate was intended to serve as a starting compound for the facile introduction of different fluorophores and small molecules via mild C-C bond formation harnessing Suzuki coupling. Following this idea, 4-iodo-BODIPY which was available in the laboratory was introduced using Pd(PPh 3 ) and Na 2 CO 3 to yield the esterprotected BAPTA-PH-BODIPY (75) with 45 % yield. Hydrolysis was achieved using LiOH following the same procedure as mentioned earlier to obtain the final BAPTA-PH-BODIPY (76). Due to the mild saponification procedure, products of satisfactory purity could be obtained after rinsing of the products with organic solvents, without further purification.

Because esters or activated ester are employed in biological studies to cross the cell membrane, 6 studies were performed on the esters (71 and 75) and the free acids (72 and 76).

3.1.2 Single crystal X-ray diffraction studies of ester protected BAPTA-BODIPYs (71 and 75)

A structural factor which is important for high fluorescence in BODIPY fluorophores, which are meso-substituted with phenyl rings, is the orthogonal orientation of the ring with respect to the dipyrromethene unit. 7 This orthogonality can be partly achieved through the incorporation of methyl groups in the α-position of the BODIPY fluorophore, which was for example used in the F 5 -BODIPY (59) introduced in chapter 2. The nature of the phenyl ring in the meso-positon also plays an important role in the orientation. A 4-bromophenyl BODIPY, for example, showed an angle between phenyl ring and diazaindecene unit of 78.9°, 8 whereas a 4-dimethylaminophenyl ring had an determined angle of 87.8° between planes. 9 On incorporating a bulky perfluorophenyl ring in the meso-position the angle becomes 90.01°, as shown in chapter 2. 3 A survey of over thirty related structures in the Cambridge Crystallographic Data Center (CCDC) revealed that the average angle between the two planes is 81.26° and can be as little as 70.7° as the least twisted 10 and essentially orthogonal with an angle of 89.85°. 11 The average deviation from the ideal planarity is 0.054 Å with a maximal deviation of 0.219 Å.

Single crystal X-ray diffraction samples of molecules 71 and 75 had been obtained from Quantum chemical calculations were performed using the MOPAC2007 (PM6 Hamiltonian, COSMO salvation model for acetonitrile). 14 In order to simplify the calculations, an aniline with a methoxy group in ortho-position was used as a representative for the BAPTA moiety, while the BODIPY fluorophore was taken in its entirety. Molecular orbitals which are potentially involved in an electron transfer reaction are highlighted in Figure 55. In both representatives (72 and 76) the HOMO-1 and the LUMO are located on the BODIPY fluorophore and show the molecular orbitals which are involved in the lowest gap opticallyactive transition. As shown in the crystal structure, the phenyl group in meso-postion to the indacene is almost perpendicular due to the steric hindrance caused by the α-methyl groups.

The angle between indacene and the phenyl ring was calculated to be 88°, which resembles the values obtained from the single crystal X-ray diffraction experiment. Regarding the HOMO for the model of BAPTA-BODIPY ( 72), it can be seen that even though almost perfect orthogonality between the planes is given, the adjacent π-system is not completely decoupled. Therefore some electronic communication between the planes is possible and could give rise to some spectral modifications to varying extents in different solvents, for example. On the other hand the model for BAPTA-PH-BODIPY (76) shows complete deconjugation of the BAPTA and the BODIPY part (figure 55, right) for HOMO-1, HOMO and LUMO levels. Thus the HOMO located on the BAPTA fragment is not affected by the transition LUMOHOMO-1 and participates as the electron donating orbital in the charge separation reaction. The twist angle here between the indacene and the phenyl spacer is estimated at 89°, which resembles the angle measured in the single crystal X-ray structure.

However, the angle between phenyl spacer and BAPTA fragment is modelled at 51° which is somewhat larger than the angle measured in the crystal structure (28°). Overall, while a similar alkoxyanilino moiety and fluorophore is present in both molecules, the absence of a phenyl spacer between both fragments leads to a more intimate coupling in the case of BAPTA-BODIPY (72), which may ultimately influence the rate of charge separation, efficiency of fluorescence enhancement upon ion binding and emission, whilst a more straightforward case may be anticipated for BAPTA-PH-BODIPY (76) which conforms more to the successful "receptor-spacer-fluorophore" concept. 15 HOMO-1 HOMO LUMO HOMO-1 HOMO LUMO The absorption spectra for the BAPTA-BODIPY esters 71 and 75 show similarly high extinction coefficients with a maximum at 501 nm, while the value for ester 75 is slightly higher due to the increased conjugated system carrying an extra phenyl ring. The band shape is characteristic for BODIPY derivatives as shown in chapter 2 and in the literature. 16 The emission spectra display a maximum at 514 nm (71) and 512 (75) with a typically small Stokes shift (∆ω = 500 cm -1 and 430 cm -1 , respectively) which are similarly low for the acids 72 and 76 (∆ω = 400 cm -1 and 440 cm -1 , respectively). Ester 71 has a low fluorescence quantum yield of 0.01 which is only 4 times higher for ester 75 (0.04). Those quantum yields are much lower compared to native BODIPY fluorophores and can be explained by efficient quenching via photoinduced electron transfer from the aniline to the fluorophore or charge transfer processes similar to the ones described for a BODIPY-crown ether derivative or a dimethylanilino-BODIPY. 17,18 Having a closer look into the emission spectra of the dimethylanilino-BODIPY it should be noted that a dual emission occurs in some solvents, among them THF, where the charge transfer emission is 47 times that of the locally excited emission. In the case of the BAPTA-BODPY ester (71) a dual emission in THF could be observed as well, however this is not the case for the BAPTA-PH-BODIPY (75) with an additional phenyl linker. The emission spectra in THF are depicted in figure 56 for comparison. The contribution of the charge transfer emission (τ = 950 ps) to the total emission could be estimated to be circa 100 times less than the locally excited emission. This second emission band disappeared in more polar acetonitrile and was not anticipated in an aqueous mixture, which will be the solvent for the calcium binding studies. The dimethylamino BODIPY by Rurack 17 showed a charge transfer band which displayed almost the same intensity as the initial S 0 S 1 emission in THF with an even higher contribution in acetonitrile. were prepared in the mentioned pseudo intracellular buffer and the concentration of Ca 2+ was adjusted using 10 mM EGTA in one buffer to result in a Ca 2+ -free solution since all free Ca 2+impurities from glassware and present in water are bound to EGTA and 10 mM CaEGTA to obtain a solution with a free Ca 2+ concentration of 39 µM. By mixing these two fluoroionophore containing buffers, the free Ca 2+ concentration was adjusted to the values given in figure 57 and figure 58 as described by Tsien. Electronic absorption spectra of 72 upon Ca 2+ -addition in pseudo intracellular buffer (bottom left). Hill plot for the spectral change in electronic absorption at 260 nm (bottom right).

For both molecules there is only a small change in the visible region of the spectra upon addition of Ca 2+ . Significant changes were observed in the UV-region of BAPTA-BODIPY Batat. 23 A short summary with essential results is described in the following pages.

Transient absorption spectra for the free acids ( solution are given in figure 59 and 60. On short timescales there is a short band which appears in the spectra of both molecules. This band was attributed to non phenomena in the solvent and was not further investigated. By fitting the gain band at 530 nm, the relaxation time of singlets was calculated to be 300 and while the gain band disappears new characteristic absorption bands rise at 570 nm, which were attributed to anion radical absorption bands of the BODIPY fluorophore which corresponds to literature data on BODIPY fluorophores. A short summary with essential results is described in the following pages.

orption spectra for the free acids (72 and 76) in buffered aqueous solution are given in figure 59 and 60. On short timescales there is a shortband which appears in the spectra of both molecules. This band was attributed to non phenomena in the solvent and was not further investigated. By fitting the gain band at nm, the relaxation time of singlets was calculated to be 300 fs for BAPTA and while the gain band disappears new characteristic absorption bands rise at nm, which were attributed to anion radical absorption bands of the BODIPY fluorophore which corresponds to literature data on BODIPY fluorophores. 21,22 Transient absorption map for BAPTA-BODIPY (72) in buffered water (λ exc = 50 in from excited singlet (right).

) shows a singlet state population which decays to give rise to radical ps. However, no evidence of triplet population from the short radicals was measured in aqueous buffer. The initial non-linear effects on the transient absorption spectrum for BAPTA-PH-BODIPY ( 76) are stronger and overlapped with the excited singlet state absorption band as can be seen in figure 60. The excited sing nm relaxes in 1.2 ps as deduced from ground state bleaching and stimulated emission signal. After a fast relaxation, anion radical absorption band of the BODIPY part still remain at 425 nm and between 600-800 nm with a decay rate of 21 ps.

Much in the way of BAPTA-BODIPY (72) no triplet states were observed for BAPTA-PH-BODIPY (76) in aqueous buffer. Consequently, radiative rate constants of 6.7 x 10 7 s -1 and 1.2 x 10 8 s -1 for 72 and 76 were obtained, respectively. These values are in good agreement to literature values for BODIPY fluorophores, 24 which are in general around 2 x 10 8 s -1 . Corresponding non-radiative rate constants are similar at 1.7 x 10 9 s -1 and 1.3 x 10 9 s -1 , respectively. The rate of charge separation (k ET ) has been calculated using equation 19 where 1/߬ corresponds to the rate of deexcitation in absence of Ca 2+ and 1/߬ మశ equals the rate of deexcitation in the presence of the ion, taking the assumption that other rate parameters are not significantly disturbed by the presence of Ca 2+ .

݇ ா் = ଵ ఛ - ଵ ఛ ೌ మశ equation 19
Electron transfer rates were calculated to be 3.3 x 10 12 s -1 for BAPTA-BODIPY (72) and 8.3 x 10 11 s -1 for BAPTA-PH-BODIPY (76) in aqueous buffer based on stimulated emission decays.

Intermediate conclusion

In conclusion, two novel BAPTA-BODIPY conjugates have been developed with efficient The receptor-spacer-fluorophore construction of 76 gives a better predictability of the behavior but ultimately the direct linkage of ionophore and fluorophore yields a more efficient switch, which will be used in following ion transfer experiments under pseudointracellular conditions in the following section.

Synthesis and spectral characterization of amphiphilic Ca 2+sensors

After the introduction of a novel water-soluble ion-sensor these properties needed to be transferred to the membrane of self-assembled vesicles. To that end, amphiphilic chemosensors based on the BAPTA ionophore were designed. This section describes the synthesis and the spectral properties of three such amphiphilic ion sensors.

Synthesis of BAPTA based amphiphilic fluorionophores bearing naphtalimide (79), BODIPY (82) and furan (97) fluorophores

The first amphiphilic Ca 2+ -sensor that was synthesized was a BAPTA-naphtalimide derivative. A previous project in the laboratory concerned the preparation of water-soluble and amphiphilic naphtalimide based ion sensors and therefore intermediates were available in the laboratory. The synthesis of fluorinated BAPTA-naphtalimide (BANIF, 79) is depicted in scheme 14. Starting from the BAPTA-aldehyde intermediate, described in the previous section, 79 was synthesized in a two step, one pot synthesis. The initial aldehyde was reacted with an amine-functionalized naphthalimide chromophore which carries an aliphatic chain on the imide functionality (77). The defining feature of the aliphatic chain is the C 8 F 17 fragment, which affords the opportunity to incorporate the resulting compound into the membrane of fluorinated vesicles.

More information about the incorporation of amphiphile molecules into fluorinated vesicle membranes is given in the following chapters. The reaction mixture containing BAPTAaldehyde (70) and fluorinated naphtalimide (77) was acidified by adding TFA in order to form an imine, which was subsequently reduced with NaBH 4 to obtain the ester protected BANIF (78). In order to increase the reactivity, hexafluoroisopropanol was chosen as solvent to give the desired amphiphilic fluorescent ion-sensor in 50 % yield. The reaction rate increasing effect of fluorinated solvents in reductive aminations had been shown by Tajbakhsh et. al.. 25 In that fashion a novel fluorescent ion-switch was designed harnessing the ionophore-spacerfluorophore format. The hydrolysis of 78 was achieved under mild conditions using LiOH in a methanol/THF mixture, which yielded the free acid (79) in sufficient purity such that after washing no further purification was needed.

The design of the second amphiphilic ion sensor is based on the previously studied BAPTA-BODIPY chemosensor. Some minor modifications had been applied to the core structure in order to render the molecule more chemically stable. The reactive β-protons which are responsible for a certain instability on silica columns and under acidic conditions were replaced by ethyl groups which have only a small effect on the absorption and emission spectra but results in a chemically more robust compound. The synthesis is shown in scheme 15.

Scheme 15: Synthesis of amphiphilic BAPTA-BODIPY (82) BAPTA-BODIPY2 (80) was synthesized according to the classical three step, one pot synthesis which was introduced in the previous section. Briefly, kryptopyrrole was reacted with BAPTA-aldehyde (70) using TFA as the reaction catalyst to form the dipyrromethane, which was subsequently oxidized to the dipyrromethene using p-chloranil. Complexation with boron trifluoride-diethyletherate was accomplished under basic conditions by adding TEA to the reaction mixture. The BAPTA-BODIPY2 intermediate (80) was obtained in 47 % yield which is almost double the reaction yield of the BAPTA-BODIPY (72) bearing reactive hydrogens in the β-position. The increase in reaction yield is mostly attributed to the much simpler purification since no additional crystallization is needed. The fluorinated chains weere introduced following a procedure of Tahtaoui. 26 The boron center of the BODIPY fragment is activated using AlCl 3 and subsequently reacted with an alcohol. In that fashion we introduced Having designed two fluorescent ion sensors which show an increase in their fluorescence intensity upon ion binding (i.e. off/on switching) a third fluorescent ion switch was envisaged with ratiometric properties. The advantage of such ion sensors is that they are unaffected by photobleaching, loading quantity or optical path length to estimate the Ca 2+ -concentration.

The design was based on the known furan derivative called "fura red" (invitrogen) which shows fluorescence emission in the visible region. The synthesis of the fura red ion-sensor was already published and patented 27 but in order to make an amphiphilic version harnessing the same properties, namely ratiometric ion binding response, the synthesis route needed to be altered in several places. The altered synthesis route is depicted in scheme 16. Decomposition of the compound could be observed after 2 -3 hours reaction time in acetonitrile. Upon switching to a non-nucleophilic amine as a base only to the dialkylated product could be isolated. The introduction of a catalytic amount of NaI to increase the reactivity of the ethyl bromoacetate improved the alkylation slightly and the triple substituted product could be isolated, while the addition of an inorganic salt led to a non-negligible amount of decomposition. In order to drive the reaction to the quadruply alkylated diamine the solvent was changed to NMP and the all-inorganic salts were omitted. In that fashion, the BAPTA intermediate ( 93) was obtained with a reaction yield of 51 %. From here on the reactions proceeded quite smoothly. The calcium complexation studies of 79, 82, and 97 was performed under pseudo intracellular conditions (100 mM KCl, 30 mM MOPS, pH 7.2), similar to those used for the watersoluble BAPTA-BODIPYs (72 and 76) described in the previous chapter, following the same titration procedure. The naphthalimide-based fluorescent Ca 2+ -switch (79) displays an increase in the fluorescence emission upon complexation of Ca 2+ . The emission maximum shifts from 550 nm in absence of the ion to 575 nm in presence of Ca 2+ . This is due to the polar excited state which exhibits a larger dipole moment than the ground state of the naphtalimide fluorophore, which leads to a sensing of the polarity of its environment. This effect has been studied and exploited in the literature to prepare, for example, ratiometric ion sensors or as a dye in cell imaging. 30,31 The red-shift of the fluorescence emission indicates a increase in the polarity of the environment which corresponds to the increasing free Ca 2+ The Stokes shift of the fluorinated BAPTA-naphtalimide (79) in presence of Ca 2+ was determined to be 5170 cm -1 and is much larger than that of the BAPTA-BODIPYs (72 and 76) presented in the previous section. Fluorescence quantum yields of 79 were determined in the presence of Ca 2+ (ɸ em = 0.026) and in its absence (ɸ em = 0.006). In absence of Ca 2+ the quantum yield is comparably low to the one measured for BAPTA-BODIPYs 72 and 76.

However, the increase in fluorescence emission upon ion complexation is lower, which was attributed to the aliphatic chain in the molecule, creating a tendency to aggregate in aqueous solution. A similarly low increase in the fluorescence emission upon Ca 2+ complexation is also expected for the amphiphilic BAPTA-BODIPY ion sensor (82).

The amphiphilic BAPTA-BODIPY derivative (82) was titrated with Ca 2+ under pseudointracellular conditions using EGTA to control the free Ca 2+ concentration. Electronic The fluorescence enhancement factor upon Ca 2+ addition was determined to be 4, calculated from the fluorescence quantum yield, which is rather low compared to its water soluble counterparts. This effect was attributed to the formation of the aggregates of this amphiphilic fluorescent switch, leading to self quenching of the fluorescence.

After linearizing the spectral change via Hill plot, a 1:1 binding with a K d of 0.724 µM has been revealed. This value is only slightly higher than the K d value obtained for BAPTA-BODIPY (72). The electronic absorption spectra of amphiphilic BAPTA-BODIPY (82) shows the typical shape of a BODIPY fluorophore. The maximum change in the absorption spectrum was observed at 265 nm. The dissociation constant, obtained via Hill plot from the absorption spectra at 265 nm, yield a K d of 0.467 µM under pseudo intracellular conditions. This value is slightly lower than the K d of 0.724 obtained from the fluorescence titration. The same observation concerning the binding affinity obtained from fluorescence and electronic absorption spectra has been made for the water soluble parent BAPTA-BODIPY (72). The third ion switch (97) was based on the ratiometric fluorescent sensor fura red (commercially available from Invitrogen). Unlike the amphiphilic fluorescent sensors 79 and 82, the fluorescence emission decreases upon Ca 2+ addition which results from a slightly lower quantum yield of the Ca 2+ -complex (ɸ em = 0.004) as compared to the non complexed form (ɸ em = 0.006). Electronic absorption and emission spectra upon Ca 2+ addition in aqueous buffer are depicted in figure 64. Fluorescence emission is observed with a rather large Stokes shift of 6790 cm -1 in the complexed and 8820 cm -1 in the non-complexed form. The emission band is broad, from 580 nm to almost 800 nm with a maximum emission at 672 nm. Linearising the spectral change at 680 nm via a Hill plot (shown in figure 64, top right) results in a K d of 0.076 µM.

Due to the additional oxygen at the BAPTA moiety, the ionophore is more electron rich and a higher binding affinity and hence a lower dissociation constant is measured for amphiphilic 32 This photoactive system has widely been exploited also for sophisticated decaging mechanisms like the simultaneous decaging of a substrate and a reporter molecule via an self-immolated cascade reaction shown in the group of Jullien. 33 The nitrobenzyl moiety was then later on exploited for the photoinduced release of divalent ions such as Ca 2+ . A first example was synthesized by Ellis-Davis et.al. 34 using an EDTA-based binding unit. The molecular structure of this Ca 2+ -decaging agent and its photoproduct is shown in Figure 65. An EDTA (8) based Ca 2+ -decaging agent has, however, the drawback that the binding affinities for Ca 2+ and Mg 2+ are both rather high. Since the cellular concentration of Mg 2+ is between 3 -4 orders of magnitudes higher than that of Ca 2+ . A set of more selective decaging agents has been developed in the group of Ellis-Davies in 1998. 35 Here the Ca 2+ binding unit is based on EGTA (9) , which has a binding affinity difference for the two divalent cations of more than 10 4 . The most notably and widely used derivative is DMNPE-4 (99) which is also depicted in figure 65. The difference in binding affinity before and after photolysis of these decaging-agents is rather high, with an affinity change of 600000 and 40000 being published in the literature as denoted in table 08. 36 This rather large change is based on the cleavage of the ion binding unit which leads to two molecules with tremendously lowered binding affinity. The mechanism for nitrobenzyl-based photocleavage is highlighted in scheme 17.

Other photoactive Ca 2+ -ejectors are based on BAPTA (10) which is a derivative of EGTA (9) bearing anilines in place of aliphatic amines. This gives rise to a slightly improved binding affinity towards Ca 2+ due to the higher electron density and an slightly improved affinity for Ca 2+ over Mg 2+ 155000 K Mg /K Ca (BAPTA, 10) and 80000 (EGTA, 9). Since the electron pair of the aniline is included in the conjugation of the aromatic ring processes like photoinduced electron transfer and it can be harnessed to create fluorescent ion switches as described in the previous section. A second advantage is the relative insensitivity of the binding affinity under physiological conditions due to the lower pKa of the anilines (pK a = 4 -6) compared to the amines in EGTA (9) (pK a = 10). This increases the conjugation of the system to the nitrobenzyl fragment which was previously separated. Due to the electron deficient nature of the nitrobenzyl chromophore, the electron density of the aniline is lowered and the molecule experiences a decrease in binding affinity from 0.145 µM to 6.3 µM, which is a 54-fold decrease. The side product during the photo-reaction is only water which makes that molecule suitable for biological studies. Tsien and Zucker used these compounds as early as 1986 to control cytoplasmic calcium with light. 38 The quantum yield for the photoreaction is rather low (0.012 -0.015) which renders this type of photo-decaging molecule inconvenient, since long irradiation times or high irradiation power is needed to release sufficient Ca 2+ which can provoke photodegradation.

The BAPTA derivative azid-1 (101) shown in figure 65 (bottom right) tackles these problems. 39 The Ca 2+ -decaging agent carries a benzofuran fluorophore on one side of the molecule much like its parent molecule fura-2 (35) which is commercially available. In the 3position of the benzofuran ring azid-1 (101), however, bears an aromatic azide. Aromatic azides undergo nitrene formation upon irradiation which can lead to a variety of products. The major photoproduct is also shown in figure 65. Passing through the nitrene state the reaction evolves towards an imminium product which carries a positive charge yielding as only side product only N 2 in the reaction pathway. This positive charge is responsible for an enormous decrease in binding affinity due to conjugation to the aniline part of the molecule. The change in binding affinity was determined to be 520-fold from 0.230 µM to essentially 120 µM. The quantum yield for the photoreaction was measured to be slightly higher than one which was attributed to the experimental error of the set up.

In recent years some progress has been made in terms of new Ca 2+ -decaging molecules. Main goal is always to create molecules with a large difference in binding affinity or a increased sensitivity towards a trigger like light which results in a higher decaging yield. In the group of del Campo Ca 2+ -decaging molecule has been developed based on the design of nitr-5 (100). 40 The A redoxactive Ca 2+ -chelator (103) has been designed by Bhattacharyya et.al.. 41 The BAPTA based binding unit is substituted in the para position of both aromatic rings with a ferrocenylethynyl-unit (see figure 66,right). First electrochemical tests have been performed which revealed a different behavior in terms of the oxidation of the aniline in presence and absence of the ion. The preliminary results of the study encouraged further investigation in electrochemical release measurements which have yet to be carried out.

However, the goal of the project was light triggered ion transfer with focus on the photoactive decaging agents. Irradiation of DM-nitrophen (98) shows a big change in binding affinity but only a rather low selectivity for Ca 2+ over Mg 2+ . While the change in binding affinity for nitr-5 (100) and its derivatives only show a moderate difference with rather low photolysis quantum yields and nitr-T was not known at the beginning of this work, the choice for the building block of the photolabile Ca 2+ -decaging agent fell to azid-1 and DMNPE-4 (99). First tests with azid-1 (101) in the laboratory showed that the photochemical quantum yield of azid-1 (ɸ = 1) actually proved problematic since light needed to be omitted entirely.

Photolysis occurred already during the transport of a azid-1 (101) sample from one room to another. Due to the high change in Ca 2+ -affinity, while retaining a high selectivity for Ca 2+ over Mg 2+ DMNPE-4 (99) was chosen as building block for the following Ca 2+ -decaging molecules in this study, even though the photochemical quantum yield was much lower than the one for azid-1 (ɸ = 0.09). The synthetic pathway largely followed the procedure published by Ellis-Davies. 35 Experimental details however were not available and therefore the synthesis was altered in several places. Reaction conditions and yields are depicted in scheme 18. The first reaction in maxima at 345 nm and 346 nm, respectively. Indeed, the difference of 1 nm falls within the error of the machine. Absorption maxima and extinction coefficients correspond well to the reported values of 350 nm and 5100 cm -1 M -1 . 36 Values for DMNPE-4 (99) and C 14 H 29 -DMNPE-4 (124) are 5110 cm -1 M -1 and 5150 cm -1 M -1 respectively as reported in table 09. The photocleavage of 99 is observed in figure 67 and is representative for both molecules since they bear the same photoactive group. In fact no evident difference could be observed between 99 and 124. Upon irradiation at 365 nm, the absorption maximum shows a hypsochromic shift towards 336 nm. Isosbestic points at 282, 315 and 355 nm indicate the clean conversion into the ketone with no other absorbing side products present.

Synthesis of Ca

Photochemical quantum yields have been determined using a ferrioxalate actinometer (see experimental section for precise procedure). 43 Briefly, a sample containing ferrioxalate was irradiated at 365 nm and subsequently the Fe 2+ -ions produced during irradiation were complexed with 1,10-phenanthroline (phen) to obtain the colored Fe(phen) 3 2+ complex.

Knowing the quantum yield for the photoreaction of ferrioxalate and the irradiation time, the amount of produced Fe 2+ per time unit and therefore the amount of incident photons per unit time could be determined by measuring the absorption of the lowest lying Metal-to-Ligand Charge Transfer absorption band of the Fe(phen) 3 2+ complex, whose molar extinction coefficient is known. Thus samples of DMNPE-4 (99) and C 14 H 29 -DMNPE-4 (124) were irradiated and the conversion into the ketone was checked via UV-Vis spectroscopy. The photochemical quantum yield was then calculated to be 0.09 (99) and 0.1 (124). The value for DMNPE-4 (99) is the same as that measured in the literature, while the quantum yield for 124 is slightly higher, which falls within the error in the measurement and may be considered essentially the same.

Ca 2+ -binding affinities could not be determined by direct titration as for the BAPTAbased fluorescent switches. Since there is no conjugation between the EGTA binding unit and the benzene ring, no discernible optical changes concomitant with complexation were observed. In order to overcome this problem an indirect determination of the K d was performed. To that end, a more concentrated solution of Ca 2+ -ejector was prepared in pseudointracellular buffer (100 mM KCl, 30 mM MOPS, pH 7.2) and CaCl 2 was been added

stepwise. The change in free Ca 2+ concentration was then monitored via a competition experiment with a fluorescent ion sensor with a known binding affinity. BAPTA-BODIPY (72) was used as the reference. 3 In figure 68 are similar it can be assumed that the change in binding affinity is equally similar upon photocleaving the Ca 2+ -recognition unit. However, the experiments have not been performed due to the lack of a Ca 2+ -sensitive electrode in the laboratory.

3.4 Ca 2+ -transfer between DMNPE-4 (99) and BAPTA-BODIPY (72) under pseudo intracellular conditions Ion transfer between photolabile ion-ejectors and a water soluble ion-acceptor has been shown in the literature for several different molecules. In most of the cases the ion transfer was used to show the actual release from a newly synthesized decaging agent. One example is the ZnCleav-2 published in the group of Burdette. 44 The molecule consists of a Zn 2+ binding unit using TPEN (N,N,N',N'-tetrakis(2-pyridylmethyl)-ethylenediamine) and a nitrobenzyl chromophore much like in the case of DMNPE-4 (99). Upon irradiation at 350 nm the Zn 2+ was released and the ion was taken up by a fluorescent Zn 2+ -switch thus increasing the fluorescence emission of the solution. Ca 2+ -transfer was shown in the group of Ellis-Davis using DMNPE-4 (99) and commercially-available Ca-orange-5N as ion detector. 36 In the following experiments the optimization of the ion transfer conditions will be carried out and

Kd = 0.032 µM R 2 = 0.992
the ion transfer between DMNPE-4 (99) and BAPTA-BODIPY (72) will be studied in aqueous solution in order to apply these conditions for the ion transfer between vesicles.

The ideal ratio between Ca 2+ -ejector (99) The titration curve using unphotolysed 99 describes a sigmoid shape due to the equilibrium between the two Ca 2+ binding species in solution. The titration with photolysed 42 shows only a small decline in fluorescence intensity with about 90 % of its initial fluorescence after the addition of 10 equivalent of photolysed ion ejector. This shows that even though the binding affinity towards Ca 2+ is greatly reduced, a certain binding of the ions remains even in the photolysed form. Between 8 and 10 equivalent of DMNPE-4 (99) the difference in fluorescence intensity is maximal. Thus, for the following ion transfer experiment 10 eq of 99 with respect to BAPTA-BODIPY (72) were used.

The photoinduced ion transfer experiment was performed in pseudo the fluorescence emission of the BAPTA of the experiment is the addition of Ca BODIPY (72) and is shown in black. The fluorescence intensity is already quite eleva (80 %) at the beginning of the experiment, which can be attributed to the impurities of Ca the glassware and the buffer solution. The addition of 5 emission to its maximum due to the uptake of the free Ca

The addition of another equivalent does not increase the fluorescence any further. The second part of the experiment consists of t Ca 2+ .decaging agent (99). Similarly to the experiment shown in figure 70, the fluorescence intensity decreases in a sigmoidal fashion due to the equilibrium between the two species until it reaches almost its minimum after the addition of 10 eq Ca 2+ ion can be considered as transferred to the ejector. The last part of the experiment is shown in blue and and consists of the irradiation of the solution at 365 the decaging agent (99) and transfer the Ca sensor (72). It is shown in figure 71 is restored resulting from the photocleavage of The maximum fluorescence emission is attained after 30 111

The photoinduced ion transfer experiment was performed in pseudo-intracellular buffer and luorescence emission of the BAPTA-BODIPY ( 72) is shown in figure 71. The first part of the experiment is the addition of Ca 2+ in the form of CaCl 2 to a solution 5

) and is shown in black. The fluorescence intensity is already quite eleva %) at the beginning of the experiment, which can be attributed to the impurities of Ca the glassware and the buffer solution. The addition of 5 µM Ca 2+ increases the fluorescence emission to its maximum due to the uptake of the free Ca 2+ by the fluorescent ion sensor ( The addition of another equivalent does not increase the fluorescence any further.

Fluorescence intensity at 512 nm of BAPTA-BODIPY (72) (λ exc = 476 nm) upon addition of CaCl ) and upon irradiation at 365 nm (blue) using a mercury lamp with monochromator on an cm distance between sample and lamp.

The second part of the experiment consists of the addition of the water soluble photolabile ). Similarly to the experiment shown in figure 70, the fluorescence intensity decreases in a sigmoidal fashion due to the equilibrium between the two species until nimum after the addition of 10 eq DMNPE-4 (99), ion can be considered as transferred to the ejector. The last part of the experiment is shown in blue and and consists of the irradiation of the solution at 365 nm in order to cleave

Conclusion

In addition to the water soluble Ca 2+ where V is the volume of the hydrophobic part of the molecule, l is the length of the hydrocarbon chain and a 0 is the effective area per headgroup. 1,2 These parameters give information on the geometrical shape of the amphiphile and thus about the tendency to form certain structures. By convention S is smaller than 1 when the hydrophilic head is curved around the hydrophobic chain and bigger than 1 when the hydrophobic part is curved around the hydrophilic head. The second case is called a reversed aggregate while the former is referred to as a normal aggregate or phase. A S value of 1 corresponds to a planar bilayer, while a value of around 1/3 indicates the formation of micelles and between 1/2 and 1 the formation of curved bilayers is possible. An example is given in figure 72. The first biomimetic vesicular structure was discovered hydrating lipids on surfaces. 3 thickness of around 4 nm encapsulating a liposomes are the ones consisting of a bilayer made of glycerophospholipid shown in figure 73. Ever since their discovery, liposomes have been studied extensively for their properties due to their resemblance to biological membranes which consist largely of phospholipids (see figure 73, 125). 5 The unique property of these capsules is the confinement of water molecules in their aqueous interior or incorporation of hydrophobic molecules in the lipid bilayer which resulted in many applications as drug delivery agents or for example as micro reactors. 6,7 Even though they are part of the cell membrane, phospholipids are not the only amphiphiles which have a tendency to self Kinitake showed in his study that a dispersion of di 126) in an aqueous medium forms uni around 3 nm. 8 From there on it became clear that a wide range of synthetic amphiphiles 117 assembled structures depended on the surfactant packing parameter (S).

The first biomimetic vesicular structure was discovered in 1964 by Bangham and Horne by 3,4 The resulting liposomes consisted of a lipid bilayer with a nm encapsulating an aqueous core. The most popular example for liposomes are the ones consisting of a bilayer made of glycerophospholipid shown in figure

Ever since their discovery, liposomes have been studied extensively for their properties biological membranes which consist largely of phospholipids (see

The unique property of these capsules is the confinement of water molecules in their aqueous interior or incorporation of hydrophobic molecules in the lipid which resulted in many applications as drug delivery agents or for example as micro Even though they are part of the cell membrane, phospholipids are not the only amphiphiles which have a tendency to self-assembly into bilayer-vesicles.

= 2 O H 2 O P O O O - O O O O R R R = C x H y O N + x = 12 -18 y = 24 -36 125
Schematic representation of a phospholipid liposome in aqueous media.

Kinitake showed in his study that a dispersion of di-n-dodecylammonium bromide (DDAB,

) in an aqueous medium forms uni-and multilamellar vesicles with a bilayer thickness From there on it became clear that a wide range of synthetic amphiphiles surfactant packing parameter (S). Vesicle membranes made of synthetic amphiphiles can be, much like liposomes, unilamellar or multilamellar. The aqueous interior typically varies between 20 -100 nm. 9 Polymerisable amphiphiles and vesicles containing polymers have been introduced in recent years and will be discussed later on. amphiphiles this effect is increased due to the higher hydrophobicity of the fluorocarbon chain and therefore exhibits a stronger character to self-organize to avoid interactions with the surrounding medium so that even single chained phosphocholines are able to form vesicles. 11 A second property which needs to be highlighted is the lipophobicity of these structures leading to a minimization of the repulsion between hydrocarbon and fluorocarbon segments. 12 Vesicles can be formed using classical methods for the preparation of liposomes. The resulting vesicles are characterized by a fluorinated core and a lipophilic shell surrounded by polar headgroups facing the aqueous medium as shown in figure 75. Fluorinated vesicles have also prepared from synthetic amphiphile-based structures. In based semi-fluorinated amphiphiles had been prepared and compared to their hydrocarbon analogous. 16 Similarly to the phosphocholine fluorinated amphiphiles the gemini-based fluorinated amphiphiles showed an increased aggregation behavior and were found to form vesicles while the hydrocarbon analogue formed entangled micelles. Another type of self-assembled structure with increased stability are vesicles consisting of block-copolymer units which will be described in the next section.

The term "polymersome" was coined in 1999 by Discher et.al. 17 and described vesicles made f a block copolymer. The block copolymers used to form those polymeric vesicles need to be of amphiphilic nature much like their low molecular weight counterparts. The length of each subunit in block copolymers is crucial for the formation of bilayers sinc structures like micelles or worm like structures are possible. 18 The properties of the containing bilayer can be easily tuned in terms of stability and permeability simply by changing the molecular weight of the polymer used. 19 Since their discovery, polymersomes, like liposomes years before, have gained considerable interest in drug delivery applications due to their ability to encapsulate different molecules like drugs, DNA or siRNA in their aqueous core or hydrophobic molecules in their membrane. 21,22 Additionally their circulation time in the human body is increased compared to liposomes and an increased stability results from a thicker membrane (3 though a lot of different methods for the preparation of polymeric vesicles are known a resemble those of liposomes, they follow mostly two main routes. The first is analogous to liposome formation via the hydration of a dried thin film with aqueous buffer yielding a polydisperse solution of different vesicles sizes. The second is the solv where the polymer is first dissolved in an organic solvent and the injected into an aqueous phase. 24,25 The stability and the mode of preparation give rise to some interesting applications in terms of multicompartmentalization since bi

Multicompartmentalized systems

Multilamellar vesicles can be considered as multicompartmentalized systems. However, it proved to impossible to do this multicompartmentalization in a controlled fashion or even with different bilayers until the year 1997. The first controlled multicompartmentalized system was reported by Zasadzinski and co consisted of small liposomes with a size of 100 with a size around 400 nm. 26 A schematic representation of the preparation process is given in figure 77. where the polymer is first dissolved in an organic solvent and the injected into an aqueous

The stability and the mode of preparation give rise to some interesting applications in terms of multicompartmentalization since bigger and more stable vesicles can be formed.

Multicompartmentalized systems

Multilamellar vesicles can be considered as multicompartmentalized systems. However, it proved to impossible to do this multicompartmentalization in a controlled fashion or even ith different bilayers until the year 1997. The first controlled multicompartmentalized system was reported by Zasadzinski and co-workers which he named vesosomes and they consisted of small liposomes with a size of 100 nm in the aqueous interior of a big A schematic representation of the preparation process is given in A schematic representation for the structure of a classical polymersome consisting of a diblock copolymer is given in Schematic representation of a vesicle formed by a block copolymer (polymersome).

olymersomes, like liposomes years before, have gained considerable interest in drug delivery applications due to their ability to encapsulate different molecules like drugs, DNA or siRNA in their aqueous core or hydrophobic molecules in their

Additionally their circulation time in the human body is increased compared to liposomes and an increased stability results from a thicker membrane (3 -4 nm). 23 Even though a lot of different methods for the preparation of polymeric vesicles are known and resemble those of liposomes, they follow mostly two main routes. The first is analogous to liposome formation via the hydration of a dried thin film with aqueous buffer yielding a ent switching method where the polymer is first dissolved in an organic solvent and the injected into an aqueous

The stability and the mode of preparation give rise to some interesting applications in terms of gger and more stable vesicles can be formed.

Multilamellar vesicles can be considered as multicompartmentalized systems. However, it proved to impossible to do this multicompartmentalization in a controlled fashion or even ith different bilayers until the year 1997. The first controlled multicompartmentalized workers which he named vesosomes and they nm in the aqueous interior of a big liposome A schematic representation of the preparation process is given in liposomes together and second it attached these liposomes to the surface of the cochleate cylinder. By adding EDTA the Ca 2+ was complexed and the cylinder was unrolled to form a single bilayer around the DLPC vesicles. This system showed several advantages in terms of cargo leakage compared to their unilamellar counterparts. In serum ciproflaxin retention was reported to increase from less than 10 min for unilamellar vesicles to over 6 h in vesosomes. 27 In the group of Bolinger, vesosomes have been prepared simply by hydrating a thin film with preformed liposomes. 28 The incorporation of liposomes with different phase transition temperatures lead to the construction of a vesosomic nano-reactor which was controlled by heating the sample above the phase transition temperature of the inner small unilamellar vesicles (SUV) to release their payload into the aqueous interior of the surrounding large unilamellar vesicle. This approach can be considered as a first example for a mimicry of cell activity.

Much effort has been invested into the fledgling field of polymeric multicompartmentalized systems, due to their higher mechanical stability 29 and their lower permeability. 30 The first multicompartmentalized system based on polymers was demonstrated by McPhail et al. 31 In this study a hybrid polymer lipid vesicle was encapsulated in the interior of an egg phosphatidylcholine liposome. A fully polymeric system was described by Chiu et.al. 32 THF/CHCl 3 as organic phase and buffered water. The primary inverted emulsion was first emulsified and subsequently added to an excess water phase, which yielded the multicompartmentalized vesicles after evaporation of the organic solvent. In the group of Lecommandoux a process which was initially invented for the almost quantitative loading of giant liposomes 33 namely emulsion centrifugation was used to prepare polymersomes in polymersomes. 34 Therefore a first set of polymersomes had been prepared using poly(trimethylene carbonate)-b-poly( L -glutamic acid) (PTMC-b-PGA) via nanoprecipitation.

The aqueous solution of (PTMC-b-PGA) polymersomes was then emulsified in an organic phase via polybutadiene-b-poly(ethylene oxide) (PB-b-PEO) and was poured over an aqueous phase. At the interface excess PB-b-PEO formed a monolayer and after centrifugation PTMCb-PGA polymersomes in larger PB-b-PEO polymersomes were obtained.

In the following study the system used by Lecommandoux and co-workers will be used to create a new kind of multicompartmentalized assembly.

Ion transfer between synthetic vesicles

This chapter will focus on ion transfer between synthetic fluorinated vesicles in the aqueous interior of a giant polymersome. Each type of vesicle (Ca 2+ -decaging and Ca 2+ -sensing) vesicles will be discussed separately and preliminary test in terms of ion transfer between vesicles will be shown.

Ca 2+ -decaging hydrocarbon vesicles preparation and ion release

Liposomes with amphiphilic photolabile Ca 2+ decaging agent (124) incorporated into the liposomal bilayer have been prepared using DOPC (1,2-di-(9Z-octadecenoyl)-sn-glycero-3phosphocholine). A thin film of amphiphilic DMNPE-4 (124) and DOPC was prepared by mixing the two components in a mixture of chloroform and methanol and slowly evaporating the solvent until a thin film was formed. Rehydration with pseudo-intracellular buffer (100 mM KCl, 30 mM MOPS, pH 7.2) yielded vesicles with the Ca 2+ -decaging agent (124) in their membrane with a final concentration of 1.5 mM DOPC and 50 µM amphiphilic DMNPE-4 (124) (formulation A). A more precise description of the experimental procedure for the vesicle preparation can be found in the experimental section. A preliminary test was carried out in order to prove that Ca 2+ can be ejected upon irradiation at 365 nm. To that end, the aforementioned formulation was doped with 5 µM water soluble BAPTA-BODIPY and 5 µM CaCl 2 in order to reproduce the ion transfer conditions determined in the previous chapter. Addition of an excess of Ca 2+ at the end of the experiment showed however that the fluorescent sensor ( 72) is capable of detecting an increase in the Ca 2+ concentration. The observed effect is most likely based on two things, first is the binding affinity of phospholipids to Ca 2+ due to the presence of an ionized phosphate group and second is the destabilisation of the lipid bilayer induced by the UV-irradiation as proposed by Uda et.al.. 35,36 Upon irradiation the phospholipid bilayer gets destabilized and breaks in several places, which gives Ca 2+ easier access to the phosphate group resulting in an decrease of free Ca 2+ and a decrease in fluorescence intensity.

After this preliminary result a different amphiphile was used for the formation of Ca 2+decaging vesicles, namely di-tetradecylammonium bromide (DTDAB (), structure shown in figure 79). Using the same preparation method as previously mentioned, formulation B has been prepared containing vesicles made of 1.5 mM DDAB, 50 µM amphiphilic DMNPE-4 (99) in pseudo intracellular buffer. The polydisperse vesicle solution has been observed with an optical microscope equiped with an irradiation source and filters for different wavelengths.

The images taken of formulation B are shown in figure 79. Polydisperse vesicles with a diameter between 200 nm and 2 µM can be observed and display a well-defined membrane without additional features. Upon irradiation between 330 and 380 nm vesicles become fluorescent after several seconds of irradiation. The appearance of fluorescence is linked to the photo-decaging of the Ca 2+ binding unit, since the resulting ketone exhibits weak fluorescence in the blue region. This experiment proves that the amphiphilic Ca 2+ decaging agent is in fact located in the membrane of the vesicles and the irradiation with UV-light does not disrupt the membrane. The substitution of the hydroxyl group was achieved in two steps without intermediate purification. The alcohol was first activated with methylsulfonic anhydride to produce an efficient leaving group and was subsequently reacted with lithium bromide to yield the heptadecafluoro-1-bromo dodecane (137) in 86 %. To obtain the final semi-fluorinated surfactant (138), heptadecafluoro-1-bromo dodecane (137) dissolved in acetonitrile in the presence of TMEDA (N,N,N',N'-Tetramethyl-1,2-ethanediamine) was stirred in a pressure tube for more than 3 days at 5 °C above the boiling point of the solvent. The surfactant (61) was obtained in 58 % yield after a double recrystalisation from a mixture of chloroform and acetone.

N + Br - N + Br - N + Br - N + Br - O O NO 2 N O O N O - - O O O O - O - OO K + K + K + K + h v N + Br - N + Br - N + Br - N + Br - O O N O N O HO O N O - O - O O O - O - O O K + K + K + K +

Ca 2+ binding affinities of fluorinated vesicles bearing 79, 82 and 97 in the bilayer

In order to see if there is a membrane-induced effect on the binding affinity, Ca 2+ -titrations were performed for amphiphilic Ca 2+ -sensors incorporated in the vesicle membrane made of the fluorinated gemini (138). To that end, three formulations of vesicles were prepared which are summarized in table 10. . Also the emission maximum can be found at shorter wavelengths. This is due to the incorporation of the vesicle membrane into the hydrophobic environment which therefore leads to a blue shift of the emission wavelength. Linearization via a Hill plot as previously described yields a straight line with a slope of almost 1 indicating a 1:1 binding of Ca 2+ to the ion-sensing vesicle. The Ca 2+ dissociation constant (K d ) was found to be 3.0 µM, which is about one order of magnitude higher than in free solution. The cationic head groups of the gemini vesicles are responsible for lowering the binding affinity since the divalent cation Ca 2+ experiences partial repulsion from the vesicle surface. Due to the different structure of amphiphilic BAPTA-BODIPY (82) and BANIF ( 79) the effect has a different impact. C 8 F 17 -BAPTA-BODIPY (82) could be less advantageously orientated on the surface and therefore display a larger decrease in binding affinity.

Formulation E has been titrated in the same fashion but the spectral change was observed in the electronic absorption spectrum due to the ratiometric nature of C 8 F 17 -furared (97). The absorption spectra in the presence of varying amounts of free Ca 2+ are shown in figure 83. The absorption spectra show an isosbestic point at 445 nm with a decreasing absorption above 445 nm and an increase between 350 and 445 nm. The spectral change at 476 nm was linearised via Hill plot, which yielded a straight line with a slope of -1.1 indicating 1:1 binding of host and guest. The extracted Ca 2+ dissociation constant (K d ) was 9.1 µM, which is also two orders of magnitude higher than in free solution (K d = 0.076 µM). Much like the amphiphilic BAPTA-BODIPY sensor (82), the amphiphilic furared (33) bears the fluorinated chain rather close to the BAPTA fragment of the molecule which locates the binding site closer to the vesicle surface and decreases the binding affinity more than for the BANIF (79) ion sensor. The change of binding affinity of Ca 2+ -sensors in liposomal membranes has already been reported for DOPC vesicles. 38 Fluorescence quantum yields have been determined for formulation C -E by comparison to fluorescein. 39 The results of the analysis are summarized in table 11. The determination of the fluorescence quantum yields in the vesicles shows that the previously mentioned low quantum yields are in fact due to aggregation of the amphiphilic Ca 2+ -sensors. In all cases the fluorescence quantum yield increases in the presence and absence of Ca 2+ . However, the

300
Kd = 9.1 µM R 2 = 0.976 Slope = -1.11
increase in emission upon Ca 2+ complexation stays rather low. The fluorescence enhancement factor calculated from the quantum yields is 1.6 for formulation C and formulation D, respectively. As observed in solution, the fluorescence quantum yield for formulation E decreases slightly upon Ca 2+ binding which can be exploited by irradiation after the isosbestic point, in that case a decrease in fluorescence intensity is caused by a lower quantum yield and a decrease in the absorption spectrum. Even though fluorescence increase is low, specific Ca 2+ -sensing supramolecular assemblies have been designed with a detectable change in fluorescence intensity. Those will be used for ion transfer experiments in the following section.

Photoinduced Ca 2+ transfer between hydrocarbon and fluorocarbon vesicles

After having prepared Ca 2+ -decaging vesicles and fluorescent Ca 2+ -sensing vesicles, ion transfer between these two populations needed to be tested.

Multicompartmentali vesicles in PB-b-PEO polymersomes

In a next step the prepared vesicle formulations needed to be incorporated into the aqueous interior of a giant polymersome. To that end the procedure published in the group of Lecommandoux 34 has been adapted for the vesicle system previously described. The multicompartmentalization process is described in figure 85. A formulation of liposomes in pseudo-intracellular buffer loaded with 130 of 380 mOsm is emulsified in toluene using b-PEO). The polymer is known to form micelles and polymersomes and has been exploited

for several studies in the literature.

water-in-oil emulsion (top left, figure 85). The emulsion was then set on top of an aqueous pseudo intracellular buffer containing 380 mOsm of glucose in order to keep the final osmotic pressure equal after centrifugation.

The excess PB-b-PEO is found at the interface between toluene and the aqueous buff 134 Multicompartmentalization of fluorocarbon and hydrocarbon

PEO polymersomes

In a next step the prepared vesicle formulations needed to be incorporated into the aqueous polymersome. To that end the procedure published in the group of has been adapted for the vesicle system previously described. The multicompartmentalization process is described in figure 85. A formulation of liposomes in ar buffer loaded with 130 mg/ml sucrose which equals an osmotic pressure mOsm is emulsified in toluene using poly(butadiene) 46 -b-poly(ethylene oxide) PEO). The polymer is known to form micelles and polymersomes and has been exploited

eral studies in the literature. 40,41 Gentle shaking produced rather big droplets of the oil emulsion (top left, figure 85).

Multicompartmentalization of synthetic amphiphile-based vesicles in the interior of giant emulsion was then set on top of an aqueous pseudo intracellular buffer containing mOsm of glucose in order to keep the final osmotic pressure equal after centrifugation.

PEO is found at the interface between toluene and the aqueous buff of fluorocarbon and hydrocarbon

In a next step the prepared vesicle formulations needed to be incorporated into the aqueous polymersome. To that end the procedure published in the group of has been adapted for the vesicle system previously described. The multicompartmentalization process is described in figure 85. A formulation of liposomes in mg/ml sucrose which equals an osmotic pressure poly(ethylene oxide) 30 (PB-PEO). The polymer is known to form micelles and polymersomes and has been exploited

Gentle shaking produced rather big droplets of the based vesicles in the interior of giant emulsion was then set on top of an aqueous pseudo intracellular buffer containing mOsm of glucose in order to keep the final osmotic pressure equal after centrifugation.

PEO is found at the interface between toluene and the aqueous buffer, forming a monolayer of block copolymer with the hydrophobic part of the molecule pointing towards the oil phase (bottom left, figure 85). In a last step, the biphasic system was centrifuged. Due to the density difference of the emulsified droplets containing 380 mOsm sucrose compared to the density of the aqueous buffer containing 380 mOsm of glucose, the emulsified droplets pass downwards through the monolayer made of PB-b-PEO. During the passage through the monolayer a second layer of polymer is enwrapped around the emulsified droplets and thus forms a bilayer, yielding giant polymersomes with the prepared vesicle solution in the aqueous interior.

In order to test the process, vesicle formulations bearing a fluorescent dye in the membrane have been prepared. Formulation H was prepared via thin film hydration using the pseudointracellular sucrose buffer to yield vesicles bearing 5 µM C 8 F 17 -BODIPY (149) in the bilayer with an amphiphile concentration of 1.5 mM. The synthesis of the BODIPY (149) will be reported later on. The molecular structure of the vesicle formulations is depicted in figure 86. Formulation I is the hydrocarbon analogue to formulation H and was prepared in the same fashion using the pseudo-intracellular sucrose buffer. Commercially available dodecyl fluorescein (139, 5 µM) was incorporated in the bilayer of hydrocarbon vesicles with an amphiphile concentration of 1.5 mM. Both formulations were extruded through a 800 nm membrane to get rid of large vesicles or aggregates. The resulting polydisperse formulations, with an average size of 308 nm (formulation H) and 398 nm (formulation I) were then used for the multicompartmentalization process and each step was followed via confocal that there are no vesicles present in the aqueous interior but all the fluorescence and therefore all the amphiphile can be found in the polymer bilayer. In fact the structures formed during the emulsion-centrifugation process are not pure polymersomes but polymer amphiphile hybrid vesicles. One characteristic of these types of structures are areas of higher and lower amphiphile content which can be seen in areas with no apparent fluorescence in the bilayer (figure 89, right). This observation has been polymer hybrid vesicles. 42 Although this was a negative result in terms of coencapsulation of the two different types of vesicle formulations these structures have a certain interest for studies of the membrane permeability which will not be further discussed here.

Because of the higher stability of the fluorinated vesicles formulations of fluorinated vesicles could coexist at the same time in the interior of the polymersome and therefore another formulation bearing a green emitting fluorescein based dye (152) in the membrane has been prepared. The synthesis of this dye is shown in chapter 5.

A schematic representation of the molecular composition of formulation J is shown in figure 89. Although this was a negative result in terms of coencapsulation of the two different types of vesicle formulations these structures have a certain interest for permeability which will not be further discussed here.

Because of the higher stability of the fluorinated vesicles 14 In order to test the ion transfer in the aqueous compartment of the giant polymersomes a solution containing 5 µM water soluble BAPTA-BODIPY (72), 5 µM CaCl 2 and 75 µM water soluble DMNPE-4 (99) in pseudo-intracellular sucrose buffer has been prepared. Ca 2+decaging agents have already been used to phototrigger the formation of hydrogels in alignate solutions 43 but the ion transfer in confined polymer compartments has not been studied.

Polymersomes containing the Ca 2+ -decaging agent and the fluorescent ion sensor in the aqueous compartment have been prepared via the emulsion-centrifugation procedure replacing the vesicle containing aqueous solution with the ion-transfer system containing ion sensor 72 and ion decaging agent polymersome is given in figure 91. During the 200 ms laser pulse at 405 fluorescence enhancement is about 5 times its initial to the ion sensor (72) during the laser pulse, the fluorescence keeps decreasing slowly, which is due to photobleaching of the BAPTA more advantageous than the co laser for the continuous irradiation. Also the Ca given moment during the experiment. After it was shown that the ion transfer in confined compartment is indeed possible the ion transfer between vesicles in the aqueous interior of the polymersome was studied. 141 polymersome preparation was taken and a fluorescence recovery after photobleaching (FRAP) experiment was conducted. The fluorescence emission in the interior of polymersome was recorded upon irradiating at 488 nm so that the Ca remains untouched. After 800 ms a light pulse at 405 nm with maximal laser intensity was ms and the irradiation of the system at 488 nm was continued. T graph from this experiment is plotted in figure 93. The fluorescence intensity in the beginning of the experiment is low due to the binding of Ca 2+ -to the decaging agent ( fluorescence emission does not increase until the laser pulse at 405 nm is given.

Average fluorescence emission of 12 independent polymersomes containing Ca sensor (72) in the aqueous interior of the polymersome. Continuous irradiation t laser pulse at 405 nm were applied and the fluorescence intensity recorded over time ms laser pulse at 405 nm the fluorescence intensity increases sharply. The fluorescence enhancement is about 5 times its initial intensity. After the ions were transferred ) during the laser pulse, the fluorescence keeps decreasing slowly, which is due to photobleaching of the BAPTA-BODIPY ion sensor (72). However this experiment is more advantageous than the continuous irradiation at 405 nm due to the use of a less strong laser for the continuous irradiation. Also the Ca 2+ -ions can be released "on demand" at any given moment during the experiment. After it was shown that the ion transfer in confined is indeed possible the ion transfer between vesicles in the aqueous interior of the polymersome preparation was taken and a fluorescence recovery after photobleaching (FRAP) experiment was conducted. The fluorescence emission in the interior of 12 nm so that the Ca 2+ -decaging agent nm with maximal laser intensity was nm was continued. The obtained graph from this experiment is plotted in figure 93 which is due to the lower quantum yield as shown before and the change in the absorption spectra which results in a lower absorption intensity at 488 nm upon Ca 2+ binding. The photobleaching is non-negligible in this experiment (10 % in 5 s) due to the increased laser power which was needed due to the lower quantum yield of the C 8 F 17 -fura red ion sensor (97). Unlike the other two cases there is only a very small decrease in fluorescence between 0 and 0.5 s noted until a similar decrease in emission due to photobleaching is noted. With formulation M there is a second suitable candidate for ion transfer studies between vesicles.

The formulation of choice, however, is formulation L due to a higher quantum yield which means the usage of lower laser powers and a slightly bigger difference in fluorescence enhancement (formulation L = 1.6, formulation M = 1.2). In the next section a formulation based on formulation L will be prepared and the ion transfer between a Ca 2+ -decaging formulation of vesicles and a Ca 2+ -sensing formulation will be studied in the aqueous compartment of a PB-b-PEO polymersome. curve where the fluorescence intensity after the laser pulse at 405 nm is in fact smaller than the initial intensity. This is due to ratio between ion transferred and photobleaching, which is unfavorable in this case. However, the fluorescence intensity increases slightly after the laser pulse, which indicates that at least partial Ca 2+ transfer is occurring. needs to be noted that the precise control of the Ca 2+ concentration is rather difficult since it depends on several parameters including the Ca 2+ impurities in the glassware, the purity of the water which can vary easily in terms of Ca 2+ and the ion impurities in the reagents used.

Therefore, a rather elevated concentration of Ca 2+ -decaging vesicles were required.

Additionally, the decrease in binding affinity once incorporated in the vesicles membrane which was determined for Ca 2+ -sensing ion acceptors (79, 82 and 97) in cationic fluorinated vesicles, most likely affects the amphiphilic DMNPE-4 (122) in the vesicle membrane as well. To that end, a higher concentration of decaging agent is needed to liberate the same . In a final experiment the rise of the fluorescence after the laser pulse at estigated in order to attribute the kinetics to either ion transfer or a decaging decaging vesicles were required.

Additionally, the decrease in binding affinity once incorporated in the vesicles membrane ) in cationic fluorinated

) in the vesicle membrane as well. To that end, a higher concentration of decaging agent is needed to liberate the same . In a final experiment the rise of the fluorescence after the laser pulse at estigated in order to attribute the kinetics to either ion transfer or a decaging process. For this experiment the same ratio between formulation O and formulation N was used as in the previous experiment but the laser pulse was kept as short as possible ( order to see a maximum of the kinetics. Assuming that the obtained rate constant is in fact connected to slow decaging of Ca DMNPE-4 based cages, it needs to be concluded that the incorporation in the vesicle membrane and the incorporation in the highly viscous aqueous compartment of the PB polymersomes somewhat slows the decaging process down. By applying the same fitting to the experiment conducted between water soluble DMNPE 150 process. For this experiment the same ratio between formulation O and formulation N was used as in the previous experiment but the laser pulse was kept as short as possible ( order to see a maximum of the kinetics. Assuming that the obtained rate constant is in fact connected to slow decaging of Ca 4 based cages, it needs to be concluded that the incorporation in the vesicle ion in the highly viscous aqueous compartment of the PB polymersomes somewhat slows the decaging process down. By applying the same fitting to the experiment conducted between water soluble DMNPE-4 (99) and formulation N a rate The slow photobleaching process after the laser pulse and continuous irradiation at 488 nm of the first 2.4 s after applying ms during the pulse to the initial intensity, gave a rate . This corresponds in the order of magnitude to the slow Ca 2+ -decaging 4 derivative (1.5 s -1 ). 47 Assuming that the obtained rate constant is in fact connected to slow decaging of Ca 2+ from 4 based cages, it needs to be concluded that the incorporation in the vesicle ion in the highly viscous aqueous compartment of the PB-b-PEO polymersomes somewhat slows the decaging process down. By applying the same fitting to

) and formulation N a rate constant of 5.83 s -1 was obtained. The later rate constant must be taken with care due to the rather long laser pulse and therefore a rather large part of the curve was extrapolated. Despite that, the rate constant is almost similar indicating that the slower release process is indeed due to the confinement in the aqueous interior of the polymersomes.

Conclusion

A new type of multicompartmentalized system has been designed, namely fluorinated cationic vesicles in polymersomes. Amphiphilic Ca 2+ -decaging agents (122) and amphiphilic Ca 2+fluorescent sensors (79, 82 and 97) have been successfully incorporated in the membrane of fluorinated gemini based vesicles and their binding affinity towards Ca 2+ determined, which showed a 1 -2 order of magnitudes lower binding than water soluble parent molecules due to the cationic environment. These ion ejecting and sensing vesicles have been successfully introduced in the aqueous PB-b-PEO polymersome compartment and the ion transfer in the aqueous interior has been achieved in a photocontrolled fashion. Fitting of the fluorescence increase during ion transfer revealed a slower decaging rate compared to literature values. In summary a new photocontrolled ion transfer system has been designed which could be the basis for photocontrolled Ca 2+ -triggered enzymatic reactions in confined microcompartments where compartmentalization is needed. This also gives the basis to photocontrolled cascade reactions in confined compartments. For example, the transfer of one type of ion to an intermediate vesicle which triggers the release of another ion which can be sensed in a third instance.

supramolecular assemblies

Introduction

Before Newton conducted his famous prism experiment it was believed that white light is essentially colourless. Nowad wavelength. In order to detect light comprising a range of different wavelengths, the human eye consists of two different types of light receptors. There are rods, which have a higher sensitivity but perceive light only in a small wavelength range, with a peak around 541 and the cones. Cones are the receptors responsible for the colour perception of the human eye but they are less sensitive, which is the reason why colours cannot be seen at nig With these CMF the tristimulus values X, Y and Z can be calculated using equations 22.

Where I(λ) is the emission spectrum of interest and

The CIE modulated tristimulus value Y to be an expression of luminosity or the brightness of the light source. That means at a given luminosity Y all chro 155 Before Newton conducted his famous prism experiment it was believed that white light is essentially colourless. Nowadays we know that white light consists of a whole spectrum of wavelength. In order to detect light comprising a range of different wavelengths, the human eye consists of two different types of light receptors. There are rods, which have a higher but perceive light only in a small wavelength range, with a peak around 541 and the cones. Cones are the receptors responsible for the colour perception of the human eye but they are less sensitive, which is the reason why colours cannot be seen at nig be divided into three different types namely ρ, ז and β cones because they display three maxima at 590 nm (orange-red), 540 nm (green) and 440nm (blue). The matching functions (CMF) representing their spectral sensitivity were determined in two independent experiments conducted by Wright 1 and Guild 2 . As such, white light as by the human, could be expressed through a mathematical formalism. These CMF showed some negative values and were therefore transformed in a way that only positive values appear. The standard observer based on these experiments was then defined by the Commission Internationale de l'Éclairage, CIE in 1931 and a graphical representation of these ̅ (λ) ݕ, ത(λ) and ̅ݖ (λ) for the standard observer are given in figure

Colour matching functions for the standard observer according to CIE1931.

With these CMF the tristimulus values X, Y and Z can be calculated using equations 22.

Where I(λ) is the emission spectrum of interest and ̅ݔ (λ) ݕ, ത(λ) and ̅ݖ (λ) the normalized CMF.

The CIE modulated tristimulus value Y to be an expression of luminosity or the brightness of the light source. That means at a given luminosity Y all chromaticities can be expressed by X Before Newton conducted his famous prism experiment it was believed that white light is ays we know that white light consists of a whole spectrum of wavelength. In order to detect light comprising a range of different wavelengths, the human eye consists of two different types of light receptors. There are rods, which have a higher but perceive light only in a small wavelength range, with a peak around 541 nm and the cones. Cones are the receptors responsible for the colour perception of the human eye but they are less sensitive, which is the reason why colours cannot be seen at night. They can and β cones because they display three red), 540 nm (green) and 440nm (blue). The itivity were determined in . As such, white light as by the human, could be expressed through a mathematical formalism. These CMF ed in a way that only positive values appear. The standard observer based on these experiments was then defined by the , CIE in 1931 and a graphical representation of these (λ) for the standard observer are given in figure

With these CMF the tristimulus values X, Y and Z can be calculated using equations 22.

(λ) the normalized CMF.

The CIE modulated tristimulus value Y to be an expression of luminosity or the brightness of maticities can be expressed by X and Z. This states for example that the colour grey has the same chromaticity as the colour white but with less intensity.

The CIE1931 colour space can then be derived from equation xx.

The CIE1931 is essentially a three dimensional space with the chromaticity in the x and the brightness in z. The graph has the shape of a horse shoe where monochromatic light can be found at the edges of the colou different wavelength and are located in the inner area of the horse shoe. White light emission can be found in the middle of the diagram shown in figure 103. 12. As one can see the definition of white light is rather broad and a wide range of coordinates can be perceived as white light. The Förster resonance energy transfer theory 5 is based on dipole-dipole interactions between two molecules, one which is the donor and one which is the acceptor. This radiationless transfer of energy occurs between molecules which are in close proximity (10-100 Å) 6 , have a spectral overlap of the donor emission spectrum and the absoption spectrum of the acceptor and lead to an decrease in fluorescence intensity and a decrease in the fluorescence lifetime of the donor. The rate of energy transfer from one fluorophore to another is dependent on the sixth power of the distance between them due to the dipole-dipole nature of the electronic excitation and can be expressed with equation 24.

ܧ = ଵ ଵାሺ ೃ బ ሻ ల equation 24
E represents the efficiency of the Förster energy transfer, R 0 the Förster radius and r is the distance between the fluorophores. The Förster distance can be calculated regarding the spectral characteristics of the fluorophores and be expressed with equation 25.

Where Ф 0 is the quantum yield of the donor in absence of the acceptor, orientation factor, n the refractive index of the solvent and

The spectral overlap of the emission spectrum of the donor and the acceptor can be calculated according to equation 26

In this formula ε A stands for the extinction coefficient of the acceptor in M normalized intensity of the donor emission spectrum and dλ is the wavelength range of the overlap. By remodelling these formulas the distance between two fluorophores can be calculated by measuring the Förster energy transfer. Vice versa knowing the distance between two fluorophores the efficiency of the energy transfer can be calculated.

One approach to obtain white light emitting polymers and polymer films has been presented by Ego et al. 7 . They report that by either endcapping, copolymerisation or covalently crosslinking the polyfluorenes with perylene fluorophores, the colour and the emission spectra of these polymers can be tuned by changing the substitution/polymer ratio. This is an example where energy transfer between two light emitting molecules is exploited to tune the emission 158

ܴ = 0.2108[ߢ ଶ ɸ ݊ ିସ ]ܬ భ ల
is the quantum yield of the donor in absence of the acceptor, the refractive index of the solvent and J the spectral overlap (Figure 104).

The spectral overlap of the emission spectrum of the donor and the absorption spectra of the . They report that by either endcapping, copolymerisation or covalently crosslinking the polyfluorenes with perylene fluorophores, the colour and the emission spectra polymers can be tuned by changing the substitution/polymer ratio. This is an example where energy transfer between two light emitting molecules is exploited to tune the emission equation 25

is the quantum yield of the donor in absence of the acceptor, К is the dipole the spectral overlap (Figure 104).

absorption spectra of the equation 26

stands for the extinction coefficient of the acceptor in M -1 cm -1 , F D is the normalized intensity of the donor emission spectrum and dλ is the wavelength range of the Schematic representation of the spectral overlap of fluorescence emission of a donor and the ling these formulas the distance between two fluorophores can be calculated by measuring the Förster energy transfer. Vice versa knowing the distance between two white light emitting polymers and polymer films has been presented

. They report that by either endcapping, copolymerisation or covalently crosslinking the polyfluorenes with perylene fluorophores, the colour and the emission spectra polymers can be tuned by changing the substitution/polymer ratio. This is an example where energy transfer between two light emitting molecules is exploited to tune the emission of the system. The first preparation of white light emitting organogels has b by Ajayaghosh and co-workers Oligo(p-phenylenevinylene)s ( acceptor in varying percentage ( occurred and was exploited to optimize the spectra for white light emission. In the group of Del Guerzo, white light emitting nano anthracene, tetracene and rubrene derivatives were mixed and their fluorescence studied.

While in solution almost complete energy transfer occurs and only the red emission of the rubrene derivative is observable, w gelation from hot DMSO. Inside the nano white light emission could be obtained. Later on, white light emitting organic liquids have been prepared in the group of Ajayaghosh those liquids is depicted in figure 106. Choosing a liquid oligo( and blending it with rubrene ( hydroxyquinolinato)aluminium (Alq be achieved. This is a very facile approach towards white light emission since the components are soluble in the organic liquid and the white light emitting ink can be obtained under completely solvent-free conditions. 159 of the system. The first preparation of white light emitting organogels has b workers 8 (see figure 105).

Molecular structure and schematic representation of the formation of white light emitting phenylenevinylene)s (141) had been co-gelated in decane with a r acceptor in varying percentage (140). As mentioned previosly, partial Förster energy transfer occurred and was exploited to optimize the spectra for white light emission. In the group of Del Guerzo, white light emitting nano-fibres have been prepared 9 . Alkyl chain functionalized anthracene, tetracene and rubrene derivatives were mixed and their fluorescence studied.

While in solution almost complete energy transfer occurs and only the red emission of the rubrene derivative is observable, white light emitting nano-fibres had been obtained after gelation from hot DMSO. Inside the nano-fibres only partial energy transfer occurs and a white light emission could be obtained. Later on, white light emitting organic liquids have e group of Ajayaghosh 10 . A schematic representation for the preparation of those liquids is depicted in figure 106. Choosing a liquid oligo(p-phenylenevinylene) ( and blending it with rubrene (144) as red emitting dye and tris(8 inium (Alq 3 , 143) as green emitting dye, white light emission could be achieved. This is a very facile approach towards white light emission since the components are soluble in the organic liquid and the white light emitting ink can be obtained under free conditions.

of the system. The first preparation of white light emitting organogels has been accomplished

Molecular structure and schematic representation of the formation of white light emitting gelated in decane with a red light emitting

). As mentioned previosly, partial Förster energy transfer occurred and was exploited to optimize the spectra for white light emission. In the group of . Alkyl chain functionalized anthracene, tetracene and rubrene derivatives were mixed and their fluorescence studied.

While in solution almost complete energy transfer occurs and only the red emission of the fibres had been obtained after fibres only partial energy transfer occurs and a white light emission could be obtained. Later on, white light emitting organic liquids have . A schematic representation for the preparation of phenylenevinylene) (

) as red emitting dye and tris(8-

) as green emitting dye, white light emission could be achieved. This is a very facile approach towards white light emission since the components are soluble in the organic liquid and the white light emitting ink can be obtained under While the previous examples made use of energy transfer between fluorophores white light emission another approach is the usage of a single white light emitting fluorophore. An example of the three different white light emitting dyes is given in figure 107 and the synthesis has been achieved by three different groups [11][12][13] . These molecules have the advantage that the emission is concentration independent The white light-emitting dyad ( creates the white light spectrum by frustrated energy transfer due to excited state intramolecular proton transfer from one hydroxytetraphenyl imidazole unit to the other. The Very few examples are known where energy transfer between the fluorophores is suppressed.

The approach of white light emitting dyes using frustrated energy transfer had been shown and another approach is the physical separation of red green and blue emitting fluorophores with the help of surfactants for example. One such system has been presented by Wang et.al. 14 and was used to produce white light emitting surface coatings. Rhodamine 110 and sulforhodamine 101 have been encapsulated in the core of poly(styrene-block-4vinylpyridine) micelles in toluene. Polyfluorene (PF) as a blue light emitting polymer has been added to that solution The mixed micelle solutions in presence of PF were then spincoated onto a substrate to cover the surface with red and green emitting micelles in a matrix of PF to form a white light emitting monolayer. A schematic representation of the system is shown in figure 108. In the current work, a new approach towards white light emitting supramolecular assemblies is shown based on nanoconfinement. So f or under pseudo-physiological conditions been described only once present white light emitting perylene bisimide vesicles at pH 9. Again, this white light emission occurs due to energy transfer from a donor molecule in the aqueous interior of the perylene vesicles and therefore molecular building blocks could not be distinguished.

In the following pages the idea of physical separation of fluorophores by encapsulating those in the membrane domain of orthogonal fluorinated vesicles is exploited, to use them as building blocks, which are subsequently sequestered in giant polymer vesicles. In doing so, white light emitting structures on the size of several µm are created, while retaini to distinguish between the R G B building blocks. The only system known to us which possesses these qualities are the white light emitting micro main difference however is the use of only two colours to pr rods and the presence of these rods in non In the current work, a new approach towards white light emitting supramolecular assemblies is shown based on nanoconfinement. So far, white light emitting vesicles in aqueous solution physiological conditions been described only once 15 . Therein the authors present white light emitting perylene bisimide vesicles at pH 9. Again, this white light nergy transfer from a donor molecule in the aqueous interior of the perylene vesicles and therefore molecular building blocks could not be distinguished.

In the following pages the idea of physical separation of fluorophores by encapsulating those membrane domain of orthogonal fluorinated vesicles is exploited, to use them as building blocks, which are subsequently sequestered in giant polymer vesicles. In doing so, white light emitting structures on the size of several µm are created, while retaini to distinguish between the R G B building blocks. The only system known to us which possesses these qualities are the white light emitting micro-rods published by Lei main difference however is the use of only two colours to prepare those white light emitting rods and the presence of these rods in non-aqueous media.

Synthesis of RGB fluorophores based on coumarin, fluorescein

The red light emitting fluorophore was based on the design of the perfluorinated BODIPY thon which features a perfluorinated phenyl ring described in chapter 2 of this thesis similar fluorophore has been published by Galangau et al. 18 The main difference in that light emitting spin coated film of red and green emitting polymer micelles and blue emitting

In the current work, a new approach towards white light emitting supramolecular assemblies ar, white light emitting vesicles in aqueous solution . Therein the authors present white light emitting perylene bisimide vesicles at pH 9. Again, this white light nergy transfer from a donor molecule in the aqueous interior of the perylene vesicles and therefore molecular building blocks could not be distinguished.

In the following pages the idea of physical separation of fluorophores by encapsulating those membrane domain of orthogonal fluorinated vesicles is exploited, to use them as building blocks, which are subsequently sequestered in giant polymer vesicles. In doing so, white light emitting structures on the size of several µm are created, while retaining the ability to distinguish between the R G B building blocks. The only system known to us which rods published by Lei et.al. 16 . The epare those white light emitting

Synthesis of RGB fluorophores based on coumarin, fluorescein

The red light emitting fluorophore was based on the design of the perfluorinated BODIPY thon which features a perfluorinated phenyl ring described in chapter 2 of this thesis 17 . A

The main difference in that BODIPY is the presence of of ethyl groups in the β-position of the core, which leads to a bathochromic shift of 28 nm (516 nm 544 nm) in the absorption and 31 nm (527 nm 554 nm) in the emission spectra due to the more electron rich nature of the compound. An advantage of the compound is the enhanced chemical stability towards chromatography which leads to improved isolated yields (33 % 38 %) and less loss of compound during purification since one purification step is sufficient to obtain the pure product. The synthesis of the red light emitting BODIPY is depicted in scheme 21.

Scheme 21: Synthesis pathway of red light emitting BODIPY (150).

After the formation of the F 5 -ethyl-BODIPY (148) a fluorinated chain bearing an heptadecafluorodecane thiol linker was introduced via S N Ar on the para position of the pentafluorophenyl ring using TEA as a base and acetonitrile as solvent. The substitution product (149) was obtained in 63 % yield after purification. The first tests on the incorporation of the fluorophore into the membrane of semi-fluorinated gemini amphiphilebased assembly were performed with this intermediate. In order to obtain a red light-emitting fluorophore, a Knoevenagel-type reaction has been performed to obtain an elongated aromatic structure in order to shift the emission wavelength towards longer wavelength. To that end, 2,4,6-trimethoxy benzaldehyde was boiled in dry toluene in presence of piperidine as a sterically hindered base. Mono and di-addition products were obtained during the reaction. By using 1.5 eq of the aldehyde and 2 eq of piperidine, monoaddition could be favoured and the final product BODred (150) was obtained in 34 % yield, which is in the range of published results on the monoaddition for Knoevenagel-type reactions on BODIPY 18,19 .

The synthesis of the green light emitting fluorophore started from commercially available fluorescein. A semi-fluorinated chain was introduced using a known procedure to form fluorescein esters 20 . C 8 F 17 -butyl bromide was reacted with fluorescein under basic conditions using K 2 CO 3 in DMF. The nucleophilic substitution occurs on the acid functionality of the molecule since the phenol is porly reactive due to the mesomeric structure of the molecule. 21 The synthesis is of the C 8 F 17 -fluorescein (152) is shown in scheme 22. The yield of the reaction was 12 % which is rather low and could be explained by the lower reactivity of long chained aliphatic compounds in polar solvents due to a coiling up of the molecule. Side products like partial alkylation of the phenols at 60°C could also participate to the lower reaction yield.

As blue light emitting fluorophore, a derivative of the commercially available pacific blue was envisaged. Due to the exorbitant price and the quantity of compound needed for functionalization it was decided to start the synthesis from commercially available difluororesorcinol (153). Following a procedure published by Weidner-Wells et al. 22 , the starting material was formylated with hexamethylenetetramine (HMTA) in strongly acid media giving 42 % of the aldehyde intermediate. In a last step a heptadecafluorodecane amine was introduced using a peptide coupling procedure with N,N,N′,N′-Tetramethyl-O-(1H-benzotriazol-1-yl)uronium hexafluorophosphate HBTU and Diisopropylethylamin (DIPEA) in THF yielding the final product pacific-C 8 F 17 (156) in 33 %.

Formation of fluorinated R G B vesicles via thinfilm hydration

Semi-fluorinated cationic vesicles loaded with red green and blue emitting dyes had been prepared using the thin film hydration procedure which is described in detail in the experimental section. The thin film was hydrated with a buffer containing 130 mg/ml sucrose, 100 mM KCl, 30 mM MOPS at pH 7.2 to obtain pseudo-intracellular conditions. The vesicle formulations had been extruded once through a 800 nm membrane in order to break large multilamellar structures. The size distribution was however rather large and vesicles of sizes ranging from 100 -2400 nm were present as shown in figure 109 While formulation R1 and B1 show a homogeneous distribution of particles, formulation G1 however shows two populations of vesicle sizes. These two populations have a maximum intensity at 180 nm and at 760 nm. Two explanations for this behaviour could be possible. A membrane during the extrusion process which was not well in place could lead two such an observation or the fluorophores themselves have an influence on the size distribution of the vesicles. For the preparation of multicompartmentalized, white light emitting supramolecular assemblies this behaviour was considered irrelevant and was not further pursued.

A summary of the fluorophore concentration in the vesicle membranes for formulation R1, G1 and B1 is given in table 13. The loading efficiency of BODIPY-red was rather low. Samples were prepared to load a total In general it could be observed that quantum yields measured with the appropriate standard, meaning a standard which emits in the same spectral range results in a slightly higher quantum yield value for the measured chromophore. This is due to the decreased sensitivity of for higher wavelength and the overcorrection of the detector used in the experiment. Mixtures of two formulations had been prepared in the ratio 1/4, 2/3. 1/1, 3/2 and 4/1 (v/v).

The coordinates of the mixed solutions are located on a straight line of the pure formulations. This is a general behaviour of mixed chromophores since the resulting colours of the mixed system are always a combination of the two emission spectra. The coordinates for the D65 ard were envisaged for the white light emitting vesicle formulation. An appropriate mixture of formulation B and G was taken (1 eq. form. B1 / 2 eq. form. G1) and doped with formulation R to obtain a white light emitting mixture which will be called ation W. The final ratio for formulation W was 1 eq form. B1/ 2 eq form. G1/ 2.5 form. R1, corresponding to a fluorophore concentration of 0.76 µM (156), 7.38

). The coordinates for the white light standard (x = 0.31271 / y = 0. Using a confocal microscope, the macroscopically observed white light emission is broken down to its blue green and red parts and single separate vesicles can be observed, where each colour corresponds to one of the formulations R1, G1 and B1. The fluorophores are well located in the vesicle membrane and no mixtures of two colours could be observed proving the rigidity and increased stability of the membrane 25 with slow amphiphile exchange rates.

The image also reveals the polydisperse nature of the formulation which is in accordance with the DLS measurements depicted in table 14. The ratio of between blue green and red vesicles corresponds well to the experimental value of 1/2/2.5. Blue vesicles can be found as the minority whereas green and red vesicles are the majority in almost equal number. In this particular picture the size of formulation G1 seems to be superior to the other two formulations.

Another mean to prove the coexistence of R1, G1 and B1 vesicles is the recording of the Fluorophores located in the inner core are well protected from solvent interactions and show therefore no decrease in their fluorescence lifetime. and B1, which implies that no energy transfer between the vesicles occurs and the fluorophores do not exchange between vesicles. Otherwise, anticipated due to spectral overlap and the fluorescence lifetime of the blue and green emission would be shortened. Figure 113 shows the spectral overlap of the absorption and emission for formulation R1, G1 and B1. It can be deduced that the parameters for energy J has been calculated using equation 26. The value for the blue green overlap is 3.43 10 1 cm -1 nm 4 which results in an estimated Förster radius (R 25. In the same fashion, the J and B1, which implies that no energy transfer between the vesicles occurs and the fluorophores do not exchange between vesicles. Otherwise, Förster energy transfer would be anticipated due to spectral overlap and the fluorescence lifetime of the blue and green emission would be shortened. Figure 113 shows the spectral overlap of the absorption and emission for formulation R1, G1 and B1.

Spectral overlap for the emission of formulation B1 and the absorption of formulation G1 (left).

Spectral overlap for the emission of formulation G1 and the absorption of formulation R1 (right).

It can be deduced that the parameters for energy transfer are given. The spectral overlap value has been calculated using equation 26. The value for the blue green overlap is 3. In order to prove that the white light emission observed in the giant polymersome is the same as that which was observe via a steady state spectra of the formu spectra was measured by a fluorescence spectrometer coupled to the confocal microscope. An overlay of the emission spectra of formulation W and the spectra measured inside the polymersome is depicted in figure 117. In the fluorescenc line) all three emissions corresponding to formulation R, G and B can be attributed without any deformation of the emission maxima. This is another indication for an absence of energy transfer between the fluorophores. The line) matches the emission spectrum of formulation W, with only minor intensity differences.

These differences are related to the different set up and excitation power used for the measurement. In order to measure a fluorescence spectrum with a confocal microscope the sample needs to be irradiated more strongly since a much smaller volume is observed and therefore much less light is emitted. 175

Confocal microscope image of a giant polymersome using an increased pinhole value (300 Blue, green and red detector channels on left and the overlay of the three detectors, tuned to the same equal nm).

In order to prove that the white light emission observed in the giant polymersome is the same as that which was observe via a steady state spectra of the formulation W, the emission

Conclusion

A new type of supramolecular white light emitting assembly has been prepared and characterized by confinement of red, green and blue emitting fluorinated vesicles in giant polymersomes. The absence of energy transfer between encapsulated vesicles has been proven and therefore the orthogonal coexistence of different fluorinated vesicles in the same compartment. Macroscopic white light emission of a formulation containing red, green and blue emitting vesicles could be transferred into a micro-environment while the supramolecular building blocks can be identified via confocal microscopy.

This approach can be useful for future work regarding cascade reactions in confined compartment were isolated building blocks in the interior are needed. Moreover fluorinated amphiphiles have several advantages over polymer formulations like precise knowledge of the size and number of molecules per vesicle, while polymers mostly consist of a size distribution. The bilayer of these vesicles is much smaller than polymeric membranes providing easier access to membrane located molecules. The unique rigidity of fluorinated vesicles could be exploited under conditions where liposomes are not stable.

The multidisciplinary approach of the thesis gave rise to a study using a wide range of techniques and comprising different disciplines of chemistry including supramolecular, photoand materials chemistry. In chapter 2 a novel perfluorinated BODIPY fluorophore (59) had been synthesized which showed an approximately three times higher photostability than commercial fluorescein and a reactivity towards biologically interesting functional groups such amines and thiols in organic solvent and aqueous DMF mixtures. The simultaneous reaction with thiols and amines present showed a 99 % selectivity for the thiol conjugate which gives rise to selective labeling applications of the amino acid cysteine in proteins. The 90-fold higher quantum yield for EtS-BODIPY (60) compared to BuNH-BODIPY (61) gives an approximate contrast of 10 4 . However, for future labeling applications the water solubility of the F 5 -BODIPY needs to be increased which can be achieved by incorporation of carboxylic or sulfonic acids on the BODIPY core in order to preserve the reactivity of the perfluorophenyl ring. Perfluorophenyl chemistry allowed the facile introduction of an azide functionality onto the aromatic ring. The azide-BODIPY (63) has been shown in some preliminary tests to be a reactive substrate for photoinduced surface patterning, on the micrometer scale, onto alkyne functionalized surfaces using a photoreducible click-catalyst (64). The quantity of azide-BODIPY (63) grafted onto the surface, was largely dependent on the irradiation power and the irradiation time, while the resolution was mostly dependent on the viscosity of the solvent. Confocal fluorescence microscopy images (FLIM) of the surface showed that in more viscous cyclohexanol a lower resolution was obtained than in toluene.

For future studies on surface patterning several parameters of the system should be investigated like the solvent viscosity dependency. Control experiments with a non photoactive precatalyst analog of (64) need to be studied in order to attribute the reactivity to the employed photocatalyst (64) as well as the formation of an apparent side product resulting from irradiation with high laser intensities.

For the principle goal of the project, the essential Ca 2+ -sensors were successfully synthesized. The spectral change at a certain wavelength is then introduced into Hill plot which is the logarithm of (F-F min )/(F max -F) where F is the fluorescence emission or the absorption at a followed by recrystallisation from chloroform, then toluene to give 347 mg (0.83 mmol, 33 % yield) of product as an orange crystalline solid.

BuNH-BODIPY (61)

F5-BODIPY (10 mg, 24 µmol) was dissolved in DMF (1 mL) and nbutylamine (20 µl, 192 µmol) was added. The reaction was followed by means of normal phase HPLC using a Kromasil SI-100 column (250 mm x 4.6 mm, 5 µm). Water was added and the aqueous phase was extracted with Et 2 O. The organic phase was washed with water and dried over Na 2 SO 4 . The solvent was removed under reduced pressure yielding the crude product (10 mg). 19 F-NMR was recorded using the crude product. The rest of the sample was dissolved in a mixture of cyclohexane / EtAc (95/5, v/v) and passed through a short SiO 2 plug to obtain 8 mg (17 µmol, 70 %) of pure product. F 5 -BODIPY (10 mg, 24 µmol) was dissolved in a Schlenk tube under nitrogen in DMF (1 mL) and diethylamine (0.1 mL, 957 µmol) was added.

The mixture was heated at 120°C overnight. Water was added and the crude product was extracted with Et 2 O and further washed with water. The organic phase was dried over MgSO 4 , filtered and the solvent was evaporated under reduced pressure to yield the crude product (6 mg). The crude sample was dissolved in a mixture of cyclohexane / Ethylacetate (95/5, v/v) and passed through a short SiO 2 plug to obtain the pure (4 mg, 8 µmol, 36 %) product. 1 H NMR (300 MHz, CDCl 3 ): δ = 6.04 (s, 2H), 4.33 (q, J = 7.1 Hz, 4H), 2.56 (s, 6H), 1.65 (s, 6H), 1.12 (t, J = 7.1 Hz, 6H). F 5 -BODIPY (300 mg, 0.638 mmol) and sodium azide (124 mg, 1.913 mmol) were dissolved in DMF (1 mL). The reaction mixture was stirred for 1 h, then diluted with brine (20 mL) and the aqueous phase was extracted with Et 2 O (3x). The organic phase was washed with brine and dried over MgSO 4 . After filtration, the solvent was evaporated under reduced pressure and the crude product was purified by column chromatography using PTE/DCM (80/20, v/v) as eluent to obtain 69 mg (0.14 mmol, 22 %) of product as a red powder. 1 H NMR (300 MHz, CDCl 3 ): δ = 3.48 (q, J = 7.02 Hz, 4H), 2.54 (s, 6H); 1.53 (s, 6H), 1.21 (t, 6H, J = 7.01 Hz). methyl-BAPTA (0.76 g, 1.26 mmol) was dissolved in a mixture of DMF (11 mL) and pyridine (0.14 ml). POCl 3 (1.0 mL, 11.1 mmol) was added dropwise and the reaction mixture stirred at room temperature overnight. Then the reaction was heated to 60 °C for 1 h. The reaction was quenched with a solution of NaOH (1 M) and the aqueous phase was extracted with DCM (3x). After washing the organic phase with brine (2x), the organic phase was dried over MgSO 4 , filtered and the solvent evaporated under reduced pressure. The crude product was triturated with isopropanol to yield 345 mg (0.55 mmol, 43 %) of pure product.

O N N O O O O O O O O O H O O N N N B N F F O O O O O O O O
triethylamine (4.0 mL) and borontrifluoride etherate (4.0 mL) were added and the mixture was stirred for two additional hours. Water was added and the product was extracted with DCM. The organic phase was dried over MgSO 4 and the solvent was evaporated. The crude product was purified by chromatography PTE/EtAc, 80:20, v/v) followed by a recrystallization in ethanol to give 0.14 g of product as an orange solid, (0.16 mmol, 26 % yield).

BAPTA-BODIPY (72)

The BAPTA-BODIPY-ester (5 mg, 6 µmol) was dissolved in a mixture of THF (1 mL) and MeOH (1 mL), then LiOH.H 2 O (5 mg, 120 µmol) and a drop of water was added and the mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure and the crude product was dispersed in ethyl acetate and centrifuged. The solvent was pipetted off, the solid dissolved in water and acidified with 1M HCl. The precipitate was dissolved in an appropiate amount of KOH solution to form the potassium salt and freeze dried to yield the title compound quasi-quantitatively. BAPTA (1.5 g, 2.5 mmol), was dissolven in dry DCM (60 ml) under argon atmosphere and cooled to 0 °C. Br 2 (0.25 ml, 4.9 mmol) in dry DCM (10 ml) was added dropwise via a syringe and the resulting solution was stirred at room temperature overnight. The reaction was quenched with 10 % aqueous NaOH, extracted with DCM and washed with water and brine. The combined organic phase was dried over MgSO 4 , filtered and the solvent evaporated. The crude was purified by recrystalization from ethanol yielding 1.23 g (1.80 mmol, 72 %) title compound. In a sealed tube, bromo-BAPTA (100 mg, 0.15 mmol), bis(pinacolato) diboron (41 mg, 0.16 mmol), potassium acetate (43 mg, 0.44 mmol) and dry DMF (2 mL) were degassed under argon during 20 min. PdCl 2 (dppf) (5 mg, 7 µmol, 5%) was added and the mixture was heated under microwave irradiation at 120°C for 15 min. The crude reaction mixture was passed through a silica plug eluting with Et 2 O and the solvent was removed under reduced pressure. The product was further purified by recrystallisation in ethanol to yield 6 as a white solid (70 mg, 65%). 1 H NMR (300 MHz, CDCl 3 ): δ = 7.32 (dd, J = 7.9 Hz, J = 1.2 Hz, 1H), 7.24 (d, J = 1.2 Hz, 1H), 6.77 -6.64 (m, 4H), 4.29 -4.27 (m, 4H), 4.17 (s, 4H), 4.15 (s, 4H), 4.05 (q, J = 7.1 Hz, 4H), 4.04 (q, J = 7.1 Hz, 2H), 2.25 (s, 3H), 1.31 (s, 12H), 1.13 (t, J = 7.1 Hz, 12H). In a Schlenk tube, BAPTA boronic ester (50 mg, 0.07 mmol), iodoBODIPY (28 mg, 0.06 mmol) and Pd(PPh 3 ) 4 (7 mg, 6 µmol, 10 mol%), were dissolved in freshly distilled THF (5 mL). An aqueous solution of Na 2 CO 3 (0.1 mL, 2 M), previously degassed by 20 min argon bubbling, was added and the mixture was heated at 65 °C overnight. The crude reaction mixture was evaporated under reduced pressure and the product was purified by column chromatography PTE/EtAc (70/30, v/v) followed by recrystallisation in EtOH to yield 38 mg (0.041 mmol, 45 %) as an orange-red crystalline powder. isopropanol (1 mL) and 5 drops of TFA were added. The reaction mixture was stirred at 80 °C for 2 h. The reaction was followed by TLC using PTE/EtAc (60/40, v/v). After cooling to room temperature (9 mg, 0.237 mmol) NaBH 4 was added and the reaction was followed by TLC using DCM/EtAc (90/10, v/v). Since the reaction did not proceed to completion, the reaction media was acidified with TFA and another equivalent of aldehyde was added.

After repeating the reduction with NaBH 4 , the mixture was filtered, evaporated and the crude product was purified by column chromatography using DCM/EtAc (90/10, v/v). 50 mg (0.039 mmol, 47 % yield) of the product was obtained as a yellow solid.

BANIF (79)

The BANIF-ester (15 mg, 12 µmol) was dissolved in a mixture of THF (1 mL) and MeOH (1 mL), then LiOH.H 2 O (10 mg, 250 µmol) and a drop of water was added and the mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure and the crude product was dispersed in ethyl acetate and centrifuged. The solvent was pipetted off, the solid was dissolved in water and acidified with 1M HCl. The precipitate was dissolved in an appropiate amount of KOH solution to form the potassium salt and freeze dried to yield the title compound quasiquantitatively.

MS (MALDI):

Calc were dissolved in DCM (30 mL) and heated to reflux for 1 h. After 30 min, some molecular sieves were added. The reaction was stopped by the addition of a saturated solution of NaHCO 3 and the aqueous phase was extracted (3x) with DCM. The combined organic phases were washed with brine (2x) and water and subsequently dried over MgSO 4 . After filtration, the solvent was evaporated to yield 1.6 g (4.5 mmol, 95 %) of product. were and heated at 90 °C in H 2 O (24 mL) for 1 h. The reaction mixture was cooled to room temperature and extracted with diethyl ether (3x) and washed with saturated Na 2 SO 3 -solution (3x). The organic phase was dried over MgSO 4 , filtered and evaporated to dryness. The crude product was purified by column chromatography using petroleum ether / ethyl acetate (80 / 20, v/v) to obtain 0.515 g of product (52 % yield). NaH (0.25 g, 10.42 mmol) was set in a round bottom flask and 20 mL THF was added at 0 °C. Trimethylsulfonium iodide (2.08 g 10 mmol) in dry DMSO (20 mL) was added dropwise at 0 °C. After a few minutes of stirring a solution of 2,4-dimethoxy-2-nitrobenzaldehyde (2.13 g, 10 mmol) in THF (5 mL) was rapidly added. The reaction was stirred at 0 °C until completed (circa 20 min) then water was added. The mixture was extracted with diethyl ether (3x) and the organic phase was washed with water and brine (5x). The organic phase was dried over MgSO 4 , filtered and evaporated.

The crude product was purified by column chromatography PTE/EtAc (70/30, v/v) to yield 1.13 g (50 %) of the epoxide. 2-(4,5-dimethoxy-2-nitrophenyl)oxirane (1.50 g, 6.66 mmol) Eu(CF 3 SO 3 ) 3 (39.5 mg, 0.066 mmol) and 2-(2-Chloro-ethoxy)ethanol (7.0 ml, 66.6 mmol) were mixed in a round bottom flask and a minimum amount of THF was added to solubilise all solids. The reaction mixture was stirred for 1 h at room temperature then separated between H 2 O and DCM. The aqueous phase was extracted with DCM (3x) and the organic phase dried over MgSO 4 . After filtration and evaporation of the volatiles, the crude product was purified by column chromatography. First two column volumes of a mixture PTE/EtAc (85/15, v/v) was used to wash of the excess alcohol then PET/EtAc was used to eluted 1.8 g (77 %) of the product, which was a light yellowish oil. To a solution of benzyl-protected DMNPE-4-ester (1.10 g, 1.46 mmol) in DCM (10 mL) was added dropwise at room temperature to a mixture of Et 2 O-BF 3 (1.86 ml, 15.9 mmol) and ethane thiol (2.22 ml, 30.8 mmol). After addition, the reaction mixture was heated at 38 °C overnight. The progression of the reaction was monitored by TLC using DCM/MeOH (95/5, v/v) Then, saturated NaHCO 3 -solution was added to stop the reaction.

The aqueous phase was extracted with DCM (3x) and the combined organic phases were washed with H 2 O (2x) and dried over MgSO 4 . After filtration, the solvent was evaporated under reduced pressure and the crude product was purified by column chromatography using PTE/EtAc (30/70, v/v) yielding 0.69 g (1.05 mmol, 71 %) of product as a light yellow oil. C NMR (75 MHz, CDCl 3 ): δ = 172.00, 171. 45, 154.41,147.19, 140.35, 132.65, 110.02, 109.26, 78.36, 70.52, 70.48, 69.00, 61.35, 60.58, 60.50, 56.62, 55.99, 55.82, 53.74, 30.82, 28.50, 17.53, 14.41, 14.36 Fine copper powder (283 mg), 1-iodoperfluorooctane (11.88 ml, 45 mmol) and 3-butan-1-ol (4.5 ml, 54 mmol) were mixed in a pressure tube and heated to 120 °C overnight. The reaction mixture was cooled to room temperature and the obtained solid was dissolved in warm chloroform and filtered hot. The solvent of the filtrate was evaporated and the crude product was recrystallized from hot hexane upon standing in the fridge. The product was obtained in 87 % (24.3 g, 39 mmol) yield. Heptadecafluorododecan-1-ol (7.8 g, 15.8 mmol) was dissolved in anhydrous THF (150 mL) and the solution was cooled in an ice bath.

Methanesulfonic anhydride (4.1 g, 23.7 mmol) and ethyldiisopropylamine (5.4 ml, 31.6 mmol) were added and the reaction mixture was stirred at 0 °C for 1 h, followed by 2 h at room temperature. The mixture was filtered and the solvent evaporated. The crude product was dissolved in acetone (60 mL) and LiBr (10 g, 115 mmol) was added and the suspension heated under reflux overnight. The acetone was evaporated and the crude product was passed through a short silica pad eluting with hexane. The organic phase was concentrated to 100 mL and the product crystallized upon standing in the freezer overnight to yield the product (86 %, 7.5 g, 13.6 mmol) as a white solid. Heptadecafluoro-1-bromo dodecane (1 g, 1.8 mmol) and tetramethylethylenediamine (127 µl, 0.85 mmol) were added to a pressure tube and the dissolved in AcCN (5 mL). The sealed pressure tube was heated at 80 °C for 3 days. The solvent was evaporated and the crude product was recrystallized two times from a 1:1 mixture of chloroform and acetone to give the product as a white solid in 58 % yield (620 mg, 0.59 mmol). The reaction mixture was heated at reflux for 6 h, cooled to room temperature and diluted with DCM. The organic phase was washed with brine (2x) and water (2x). After drying over MgSO 4 , the solution was filtered and evaporated to dryness. The crude product was purified by column chromatography using PTE/DCM (30/70, v/v) as eluent to obtain a blue solid 12 mg (0.011 mmol, 34 %). In a Schlenk tube, 2,4-difluororesorcinol (100 mg, 0.68 mmol) was dissolved in TFA (1 mL) and hexamethylenetetramine (190 mg, 1.36 mmol) was slowly added under stirring. The tube was placed under a N 2 -atmosphere and heated to reflux overnight. The reaction mixture was cooled and H 2 SO 4 (50 %, 2 mL) was added and stirred for 2 h. The acidic aqueous phase was extracted EtAc (3x) and the combined organic phases were washed with HCl (1 N, 3x) and water (1x). The organic solution was dried over MgSO 4 , filtered and the solvent was evaporated under reduced pressure to yield 50 mg (0.287 mmol, 42 %) product as a white solid. Pacific blue (155) 8 2,4-Dihydroxy-3,5-difluoro benzaldehyde (50 mg, 0.287 mmol) and malonic acid (32 mg, 0.316 mmol) were dissolved in methanesulfonic acid (2 mL) and the reaction mixture was stirred at room temperature overnight. The solution was diluted with brine and extracted with a THF/EtAc mixture (3x).

The organic phase was dried over MgSO 4 , filtered and evaporated. The crude product was purified by column chromatography using DCM/MeOH/Formic acid (80/20/1, v/v/v) as eluent to obtain 40 mg (0.165 mmol, 53 %) of pure product.

Photocontrolled biomimetic communication between molecules and nanosystems in confined compartments

The thesis focuses on the study and design of novel photoactive molecules and their

Image 01 :Image 02 :

 0102 photo-actif DMNPE (1-(4,5-dimethoxy-2-nitr

Alq 3 =

 3 tris(8-hydroxyquinolinato)aluminium ATP = adenosine triphosphate BAPTA = 1,2-bis(o-aminophenoxy)ethane-cAMP = cyclic adenosine N,N,N',N'tetraacetic acid monophosphate DDP = di-n-dodecyl phosphate DDAB = di-n-dodecylammonium DHP = di-n-hexadecyl phosphate DIPEA = N,N-Diisopropylethylamine DLPC = 1,2-dilauroyl-sn-glycero-DMNPE = dimethoxynitrophenyl 3-phosphocholine DMPC = 1,2-dimyristoyl-sn-glycero-DOAB = di-n-octadecylammonium 3-phosphocholine bromide DOPC = 1,2-di-(9Z-octadecenoyl)-sn-glycero-DOPS = dioleylphosphatidylserine 3-phosphocholine DPPE = 1,2-dipalmitoyl-sn-glycero-Dppf = [1,1-bis(diphenyphosphino)-3phosphoethanolamine ferrocene] DTDAB = di-tetradecylammonium bromide EDTA = Ethylenediaminetetraacetic acid EGTA = (ethylene glycol tetraacetic acid HBTU = N,N,N′,N′-Tetramethyl-O-(1Hbenzotriazol-1-yl)uroniumhexafluorophosphate HEEDTA = N-(2-hydroxyethyl) -HMTA = Hexamethylenetetramine ethylenediamine N,N',N'-triacetic acid MOPS = 3-[N-morpholino]propanesulfonic PB-b-PEO = polybutadiene-bacid poly(ethylene oxide) PGE = polyglycerol PMMA = Poly(methyl methacrylate) Poly(AAc-co-DSA) = Poly(acrylic acid-co-PTMC-b-PGA = poly(trimethylene distearin acrylate) carbonate)-( L -glutamic acid) TMEDA = N,N,N',N'-Tetramethyl-1,2-b-poly TPEN = (N,N,N',N'-tetrakis(2

Figure 01 :

 01 Figure 01: Schematic representation of the three structural forms of calmodulin. (a) apo-CaM structure. (b) Ca-

Figure 02 :

 02 Figure 02: Schematic representation of ion transfer inside a cell and the adopted approach to photo-guided ion transfer in artificial microcapsules.

Figure 03 :

 03 Figure 03: left: Valinomycin molecular model binding a potassium ion, right: chemical structure of valinomycin.

1

 1

Figure 06 :

 06 Figure 06: pH-triggered ion translocation left: molecular syringe translocating a silver ion from the crown ether cavity to the bis(ethoxyethoxy) binding site, right: Cu 2+ translocation as proposed by Fabbrizzi and co-workers.

Figure 07 :

 07 Figure 07: Example of an ion-induced ejection of 1,5-diaminopentane upon the addition of ZnI 2 (13) (left) and sodium ejection from a dicrown ether (14) upon the addition of Hg 2+ (right).

Figure 08 :

 08 Figure 08: Excited state through a n π* transition into a singlet excited state (S 1 )

Figure 09 :

 09 Figure 09: Electronic states

  has absorbed a photon and passes to the excited-state several different deexcitation processes are possible. The Jablonski-Perrin diagram 40 visualizes all possible deexcitation processes in a graphical manner. A simplified version of this diagram is depicted in figure 10.

Figure 10 :

 10 Figure 10: Simplified version of the Jablonski-Perrin Diagram

4

 4 photoprocess can be calculated according to equation 03.ɸ ி =

Figure 11 :

 11 Figure 11: trans-cis isomerisation of an azobenzene molecule (15) applying an external trigger like light or temperature.

Figure 12 :

 12 Figure 12: ring open-close isomerisation of an perfluoro dithienylethene (16) applying light as an external trigger.

Figure 13 :

 13 Figure 13: Phototriggered ejection of Zn 2+ irradiation of an phenolic Schiff-base-Zn 2+ complex (17) via UV irradiation.

Figure 14 :

 14 Figure 14: a) Azobenzenophane (18) crown ether synthesized by Shinkai and co-workers. Reversible ion release / uptake upon irradiation or thermal relaxation. b) Photocontrolled proton ejector based on an azobenzene chromophore (19).

Figure 15 :

 15 Figure 15: Reversible Ca 2+ from aza-crown ether unit by modulation of the electron density of the amine using a dthienylethene chromophore in a non-competitive solvent.

Figure 16 :

 16 Figure 16: Principles of photoinduced electron transfer (PET) of an off

Figure 17 :Figure 18 : 22 based

 171822 Figure 17: Principles of photoinduced electron transfer (PET) of an off

  -selective ion sensors are of particular interest. When elaborated with ligating sites, receptors bearing an aromatic amine are on fluoroionophore by oxidative PET. sensors, the electron pair of an aromatic or an aliphatic amine is involved in the quenching mechanism. Several examples which are sensitive to different cations such as Zn 2+ , A structural simple example of a fluoroionophore for potassium is based on an azacrown ether linked via a methylene spacer to an anthracene fluorophore developed in the group of de Silva 65 (21, ent up to 47 times its initial intensity on fluorescent ion switch was crown ether where all the oxygens are eating therefore a selectivity towards Zn 2+ . The advantage of the system is that it could be used in aqueous solution but needs precise pH which also leads to a quenching of the PET. If the fold could be achieved and Na + (21) developed by de selective ion sensors are of particular ring an aromatic amine are especially suited for fluorescent ion sensing under physiological conditions. Selective ion sensors for Ca 2+ are in general based on the BAPTA binding unit described earlier. In principle to obtain a fluoroionophore literally thousands of combinations between ionophore and fluorophore are possible. Two Ca 2+ -selective probes are highlighted in figure 19.

Figure 19 :

 19 Figure 19: Ca 2+ off-on fluoroionophores based on the BAPTA binding unit.

Figure 20 :

 20 Figure 20: Fluoroinophores based on the BODIPY fluorophore with an alkali and alkaline earth metals sensitive azacrown (25) (left) and Ca 2+ -selective BAPTA-binding site (26) (right).

Figure 20

 20 Figure 20 on the right side shows a series of BAPTA-BODIPY fluoroionophores with different substitution patterns which are linked together via a 3-propanamide linker(26).72 The fluorescence is greatly quenched due to PET in aqueous medium. The binding affinities towards Ca 2+ are in the range of 200 nM. Upon Ca 2+ binding greatly enhanced fluorescence was obtained with no notable spectral shift due to the pH insensitivity of the fluorophore. The first example of fluorescent BAPTA sensors excitable in the visible light region were developed by Tsien.73 In figure 21 some chemosensors from the rhod and the fluo series are depicted. The dyes were developed to overcome the problems which are imposed by UVexcitable dyes such as UV damage to cells upon irradiation, provoking autofluorescence from pyridine nucleotides and the lack of simultaneous application of Ca 2+ decaging agents with UV excitable chemosensors. The latter reason is most important for the goal of the work in hand. These sensors, unlike their UV light-excitable counterparts, can be used in confocal fluorescence microscopy, two-photon excitation microscopy, and total internal reflection microscopy.74 The first generation of rhod(27-1) and fluo (28-1) chemosensors bearing a hydroxyl group in the meta-position of the aniline showed strong pH dependency in the physiological range. This problem was overcome by omitting the hydroxyl group leading to the more potent rhod-2 (27-2) and fluo-2 (28-2) dyes.

Figure 21 :

 21 Figure 21: Visible light excitable Ca 2+ -selective BAPTA based fluoroionophores developed by Tsien.

Figure 22 :

 22 Figure 22: Photoinduced electron transfer principle of an "on

Figure 23 :

 23 Figure 23: on-off fluoroinophores for proton sensing based on a triphenyl pyrazoline (29) (left) and for mercury sensing based on a dansyl-substituted calyx-4-arene (30) (right).

Figure 24 :

 24 Figure 24:

Figure 24 :

 24 Figure 24: Energy profile of an electron transfer reaction

Figure 25 :

 25 Figure 25: Schematic representation of a fluorophore

Figure 26 :

 26 Figure 26: Spectral displacements of PCT sen donating or an electron-withdrawing group.

Figure 27 :

 27 Figure 27: Crown ether based PCT ion sensors.

Figure 28 :

 28 Figure 28: BAPTA based Ca 2+ -selective PCT sensors.

Figure 29 :

 29 Figure 29: Multi-adressable molecular systems based on an ion and proton binding unit linked to a fluorophore.

Figure 30 :

 30 Figure 30: Red light emitting BAPTA based fluorinophore bearing a x-rhodamine fluorophore (39).

Figure 31 :

 31 Figure 31: molecular structure of the BODIPY core compound (40)

Figure 32 :

 32 Figure 32: Possible post-functionalization of the BODIPY skeleton.

( 41 ) 11 Scheme 04 :

 411104 Scheme 04: Preparation of a potassium sensor by S n Ar on a chlorinated BODIPY fluorophore.

Figure 33 :

 33 Figure 33: Examples for S E,Ar in β-position of BODIPY

Figure 34 :

 34 Figure 34: Ball and stick representation of the X

Figure 35 :

 35 Figure 35: Reaction scheme and HPLC traces showing the conversion of F5-BODIPY (59) to EtS-BODIPY (60) (left) and to BuNH-BODIPY (61) (right) after different reaction times recording at 520 nm.

position of 59 .

 59 F-NMR spectrum of the crude reaction mixture displayed fluorine signals at -147.6 and -161.6 ppm indicating the clean substitution in the 4-The parent molecule did not react with diethyl amine at room temperature.

Figure 36 :

 36 Figure 36: left: Direct comparison of the reactivities of F 5 -BODIPY (59) with thiol and amine conjugates; right: HPLC-trace of the reaction of 59 with a mixture of ethane thiol and butyl amine recorded at 520 nm.

Figure 37 :

 37 Figure 37: Progression of the reaction forming EtS-BODIPY (60) in a mixture of DMF:H 2 O (1:1, v/v) using 5 eq of ethane thiol and 8 eq of butyl amine.

Figure 38 :

 38 Figure 38: Absorption and emission spectra of F 5 -BODIPY (59) in air equilibrated THF (left) and of the substitution products EtS-BODIPY (60), BuNH-BODIPY (61) and Et 2 N-BODIPY (62) (right). Emission has been recorded upon irradiation at 465 nm.

Figure 39 :

 39 Figure 39: Time-correlated single photon counting spectrometry and fitting of the fluorescence lifetime for 59 (left). Residuals of fit in terms of standard deviation for the fitting of the lifetime (right, chi-square: 1.5).

Figure 41 :

 41 Figure 41: Reaction scheme for photodegradation.

equation 12 Byequation 14

 1214 taking in account equation 12, the changes of the ground state population or the number of molecules in the ground state (N g ) in time can be expressed as shown in equation 13. Substituting equation 09 in equation 13 and having in mind the photon flux from equation 10, one obtains for slowly varying steady-state absorption equation 14 Initial condition is N G = N at the beginning of the experiment (t=0) and ܿ of initial solution is small, the exponent in the Taylor series can be developed 10 ௫ = ݁ ௫ଵ = 1 + .+01݈݊ܽݔ .. equation 15 and after insertion into equation 14, equation 16 can be formulated. That corresponds to an exponential relaxation of the ground state population or the steady state absorption with a time constant expressed in equat Thus, the quantum yield of the irreversible photoreaction (photodegradation) can be found from the exponential kinetics of a steady state absorption decay upon irradiation as equation 18. The quantum yields of photodegradation for molecule equilibrated THF by irradiating a stirred solution of the molec at 504 and 512 nm. Photodegradation graphs with corresponding fitting are depicted in figure 42.

Figure 42 :

 42 Figure 42: Left: Changing absorption at 464 nm accompanying the photodegradation of fluorescein i equilibrated ethanol as a function of irradiation time, 515 nm accompanying the photodegradation of F irradiation time, λ exc = 512 nm (P=3.9 mW).

  an exponential relaxation of the ground state population or the steady state absorption with a time constant expressed in equation 17 ݎ = ௩ ௐఌொଵ Thus, the quantum yield of the irreversible photoreaction (photodegradation) can be found from the exponential kinetics of a steady state absorption decay upon irradiation as ܳ = ௩ ௐఌଵ The quantum yields of photodegradation for molecule 59 -62 were then determined in air equilibrated THF by irradiating a stirred solution of the molecule under continuous irradiation nm. Photodegradation graphs with corresponding fitting are depicted in figure absorption at 464 nm accompanying the photodegradation of fluorescein i equilibrated ethanol as a function of irradiation time, λ exc = 504 nm. (P=3.5mW). Right: Changing absorption at 515 nm accompanying the photodegradation of F 5 -BODIPY (59) in air-equilibrated acetonitrile as a function of nm (P=3.9 mW).

equation 16 That 59 Left:

 1659 corresponds to an exponential relaxation of the ground state population or the steady equation 17 Thus, the quantum yield of the irreversible photoreaction (photodegradation) can be found from the exponential kinetics of a steady state absorption decay upon irradiation as denoted in equation 18 were then determined in airule under continuous irradiation nm. Photodegradation graphs with corresponding fitting are depicted in figure 200 250 Changing absorption at 464 nm accompanying the photodegradation of fluorescein in air-Changing absorption at equilibrated acetonitrile as a function of

  Measuring in time-tagged time resolved (TTTR) mode, emission trajectories and decay times could be obtained. The total number of photons and emission duration before photobleaching, as well as the peak emission intensity of each single molecule after removing the contribution of the background, which consisted of a weak PMMA emission and instrumental background, were measured. Each fluorescence lifetime shown in figure 43 corresponds to the longest lifetime resulting from a biexponential decay.

Figure 43 :

 43 Figure 43: Single molecule emission of 59 in PMMA (λ ex =488 nm, λ em > 500 nm): (a) emission trajectory of a single molecule (number of photons detected per ms); (b-d) distribution for 82 single molecules of: the total duration of emission before photobleaching (b); the decay time (c); correlation between the decay time and the average number of counts emitted before photobleaching (d).

Figure 44 :

 44 Figure 44: Histogram of the total number of photons collected before photobleaching for 82 molecules of F 5 -BODIPY (59).

Figure 45 :

 45 Figure 45: Photoinduced patterning of a PEG-based hydrogel onto an azide-functionalized polypropylene using a photomask.

  Scheme 08: Synthesis of azide-BODIPY(63)

Scheme 09 :

 09 Scheme 09: Molecular structure of the photoreducible copper(II)-tren complex [Cutren(CH 2 PhtBu) 3 ](kt) 2, 64. kt = ketoprofenate.

Scheme 10 :

 10 Scheme 10: Click-reaction between azide-BODIPY (63) and dodec-1-yne catalysed by photolatent copper catalyst [Cutren(CH 2 PhtBu) 3 ](kt) 2 (64).

Figure 46 :

 46 Figure 46: Absorption and emission spectra of the click-product in THF (λ exc = 510 nm) (65).

Figure 47 :

 47 Figure 47: Schematic representation of the surface patterning procedure using a solution containing 50 mM azide-BODIPY (63) and 1 mM [Cutren(CH 2 PhtBu) 3 ](kt) 2 (64) in toluene.

Figure 48 :

 48 Figure 48: Scheme of the confocal microscopy setup used to observe the surfaces.

Figure 49 : 64 Scheme

 4964 Figure 49: Left: Confocal microscopy image (800 x 800 surface (LD) fresh from the box. Right: Confocal microscopy image of the cleaned surface after irradiating at

Figure 50 :

 50 Figure 50: Left: A summary of the experiment conducted, indicating the number of passages, laser power and duration of irradiation time per/pixel at 375 after washing (LD surface). Images obtained by scanning the surface with the observation setup shown in figure 48 with a speed of 1.248 ms/pixel.

65 BODIPY ( 63 )

 6563 and [Cutren(CH 2 PhtBu) 3 ](kt) 2(64) in toluene while covering halfThe first observation is that the fresh LD-surfaces show almost no fluorescence background resulting from impurities or defects on the surface, see figure49left. However, the LD surface that was subjected to irradiation shows a clear stepwise increase of the intensity with higher fluorescence on the irradiated side and a side with lower fluorescence corresponding to the position of the photomask. It should be noted that a certain fluorescence intensity is also irradiated part of the surface due to the incubation with fluorophore. The difference in fluorescence is nevertheless specific.surface was used and covered with a toluene solution of azide ). In figure50the conducted experiment is described on the left side and the obtained fluorescence lifetime imaging microscopy (FLIM) image is shown on the right. nm with a power of 400 au (corresponding to nW +/ "write" squares with a size of 20 x 20 µm, on the surface. Four patterns were produced by scanning the same area several times with the same constant speed of 1.2 ms/pixel of 100 of a constant CW power of 400 nW. The scanning pattern is shown in figure50The brighter canner parking. The FLIM images on the right side of figure50show the fluorescence intensity in terms of brightness as well as the fluorescence lifetime of the emitted light. First it is seen that an increased the number of passages results in a brighter fluorescence emission with a maximum at 3 passages. Left: A summary of the experiment conducted, indicating the number of passages, laser power and duration of irradiation time per/pixel at 375 nm. Right: Confocal microscopy image of the c after washing (LD surface). Images obtained by scanning the surface with the observation setup shown in figure A2 2x400au@1.2ms A4 4x400au@1.2ms ) in toluene while covering half surfaces show almost no fluorescence background resulting from impurities or defects on the surface, see figure 49 left. However, the LDncrease of the intensity with higher fluorescence on the irradiated side and a side with lower fluorescence corresponding to the position of the photomask. It should be noted that a certain fluorescence intensity is also the surface due to the incubation with fluorophore. The surface was used and covered with a toluene solution of azide iment is described on the left side and the obtained fluorescence lifetime imaging microscopy (FLIM) image is shown on the right. au (corresponding to nW +/-5%) was used to µm, on the surface. Four patterns were produced by ms/pixel of 100 mm nW. The scanning pattern is shown in figure 50 The brighter canner parking. The FLIM images on the right side of figure 50 show the fluorescence intensity in terms of brightness as well as the fluorescence lifetime of the emitted light. First it is seen that an increased the number of passages results in a brighter Left: A summary of the experiment conducted, indicating the number of passages, laser power and nm. Right: Confocal microscopy image of the conducted experiment after washing (LD surface). Images obtained by scanning the surface with the observation setup shown in figure

  (left). The obtained image showing intensity and fluorescence lifetime is depicted in figure 51 (right).

Figure 51 :

 51 Figure 51: Left: A summary of the experiment conducted indicating laser power, wavelength and duration of irradiation time per/pixel. Right: Confocal FLIM image of the conducted experiment after washing (HD surface).Images obtained by scanning the surface with the observation setup shown in figure48with a speed of 0.600 ms/pixel.

Figure 52 :

 52 Figure 52: Left: Fluorescence intensities of points created with 400 intensities of points created with 10000 observation setup shown in figure 48 with a speed of 0.600 ms/pixel.

  nW laser power. Right: Fluorescence anning the surface with the nm irradiation the fluorescence intensity increases nal increase is noted for the nW. show an increase until 6 s of irradiation, volve the diffusion of catalyst mM [Cutren(CH 2 PhtBu) 3 ](kt) 2

Figure 53 :

 53 Figure 53: Left: A summary of the experiment conducted indicating laser power, wavelength and duration of irradiation time per/pixel. Middle: Confocal fluorescence microscopy image of the conducted experiment after washing (HD surface). Right: Fluorescence emission spectra obtained from surface top right, bottom left and bottom right. The colours of the curve correspond to the colours of the squares shown in the image.

2 , 3

 23 Aldehyde (70) was reacted with 2,4-dimethyl pyrrole to afford the dipyrromethane intermediate,which was subsequently oxidized to dipyrromethene using p-chloranil. Complexation with BF 3 -Et 2 O lead to the desired product(71), after column chromatography and subsequent crystallization from ethanol with a yield of 26 %. The saponification of the esters under mild conditions, using an excess of LiOH, yielded the ion sensitive BAPTA-BODIPY fluorescent switch(72).

Scheme 13 :

 13 Scheme 13: Synthesis of BAPTA-PH-BODIPY(76)

  ethanol and the determined structures are shown in figure 54. The crystal structure confirms the orthogonal arrangement of phenyl plane in the ester protected fluorionophores 71 and 75 which is imposed by the substituents of the BODIPY subunit. The dihedral angle between BODIPY and phenyl fragment planes is 87.2° for 71 and 85.8° for 75, respectively. The phenyl rings in the biphenyl unit of BAPTA-PH-BODIPY ester (75) are slightly tilted with a dihedral angle of 28.0°.

Figure 54 : 13 3. 1 . 3

 541313 Figure 54: Stick representation of the single crystal X-ray structures of BAPTA-BODIPY ester (71) (a) and BAPTA-PH_BODIPY ester (75) (b), whose crystals were obtained from ethanol. Atoms and corresponding colours are: carbon (grey), oxygen (red), nitrogen (blue), boron (pink) and fluorine (green).

Figure 55 :

 55 Figure 55: HOMO-1, HOMO and LUMO orbitals of model compounds for molecules 72 (left) and 76 (right) calculated by MOPAC 2007.

Figure 56 :

 56 Figure 56: Emission spectra of BAPTA-BODIPY ester (71) and BAPTA-PH-BODIPY ester (75) in air equilibrated THF (λ exc = 475 nm).

3. 1 . 5

 15 Ca 2+ complexation studies of BAPTA-BODIPYs (72 and 76) The effective dissociation constants (K d ) of the calcium complexes of fluorionophores 72 and 76 were determined in an aqueous buffer under pseudo-intracellular conditions (30 mM MOPS (3-[N-morpholino]propanesulfonic acid), 100 mM KCl at pH 7.2) by UV-Vis and fluorescence spectroscopy as a function of Ca 2+ . The precise titration procedure can be found in the experimental section. In short, a solution containing 1-10 µM of Ca 2+ -sensor 72 or 76

Figure 57 :

 57 Figure 57: Fluorescence emission spectra of BAPTA-BOIDPY 72 upon Ca 2+ -addition in pseudo intracellular buffer (λ exc = 476 nm) (top left). Hill plot for the spectral change in fluorescence emission at 515 nm (top right).

( 72 )

 72 , due to the perturbation of the alkoxyanilino moiety upon complexation, with a maximal change at 260 nm. BAPTA-PH-BODIPY(76) shows two significant bands which alter upon Ca 2+ addition with a maximal change at around 260 nm and a second band with a maximal change at 316 nm. The second band is attributed to the presence of the additional phenyl spacer between fluorophore and the BAPTA fragment. These spectral variations allowed the calculation of the ground-state dissociation constant (K d ). Linear fitting of the spectral change via Hill plot (figure 57 and 58 bottom right) which is the logarithm of the spectral change plotted over the logarithm of the free Ca 2+ concentration, yields a straight line for mono-ionic binding. The slope of the resulting linear fitting indicates the order of the complex.

Figure 58 :

 58 Figure 58: Fluorescence emission spectra of BAPTA-PH-BODIPY (76) upon Ca 2+ -addition in pseudo intracellular buffer (λ exc = 476 nm) (top left). Hill plot for the spectral change in fluorescence emission at 509 nm (top right). Electronic absorption spectra of 76 upon Ca 2+ -addition (bottom left). Hill plot for the spectral change in electronic absorption at 316 nm (bottom right).

3. 1 . 6

 16 Time-resolved transient absorption studies of BAPTA-BODIPYs (72 and 76) Transient absorption spectroscopy provides the possibility to follow the evolution of the excited state of a molecule in the first few pico to nanoseconds after excitation. Since the BODIPY triplet absorption spectra spectroelectrochemical study of the BODIPY radical anion formation has been carried out, it is now possible to disentangle the electron transfer pathway. The transient absorption measurements have been carried out in collaboration with the university of Bordeaux. The obtained results are shown in great detail in the thesis of Dr.

Figure 59 :

 59 Figure 59: Transient absorption map for BAPTA 540 nm of radical grow-in from excited singlet (right).

Figure 60 :

 60 Figure 60: Transient absorption map for molecule BAPTA-PH-BODIPY (76) in buffered water (λ exc = 500 nm) and kinetics of radical grow-in from excited singlet (right) at 540 nm.

Figure 61 :

 61 Figure 61: Transient absorption maps for BAPTA-BODIPY (72) (left) and BAPTA-PH-BODIPY (76) (right) in the presence of Ca 2+ in aqueous buffer (ʎ exc = 500 nm).

  Ca 2+ binding-induced fluorescence enhancement. A different behavior could be observed in aprotic solvents like THF compared to protic water. The electron transfer is several times faster in water and unlike in the organic solvent, no triplet formation from the chargeseparated state has been obtained. As a result of the ion-binding induced blocking of the PET fluorescence enhancements of 122 and 23 fold are measured for BAPTA-BODIPY(72) and BAPTA-PH-BODIPY (76) respectively making them effective fluorescent chemosensors.

Scheme 14 :

 14 Scheme 14: Synthesis of amphiphilic Ca 2+ -chemosensor 79.

C 8 F

 8 17 -ethanol onto the boron by substituting the fluorine atoms. The ester protected C 8 F 17 -BAPTA-BODIPY(81) was obtained in 26 % yield which is lower than the reaction yields reported in the paper due to partial hydrolysis of the esters judged by TLC. The final molecule(82) was then obtained quantitatively after ester hydrolysis via LiOH in a MeOH / THF mixture after a simple washing with organic solvents.

Figure 62 :

 62 Figure 62: Fluorescence emission spectra of BANIF (79) upon Ca 2+ -addition in pseudo-intracellular buffer (λ exc = 450 nm) (top left). Hill plot for the spectral change in fluorescence emission at 575 nm (top right). Electronic absorption spectra of 79 upon Ca 2+ -addition (bottom left). Hill plot for the spectral change in electronic absorption at 245 nm (bottom right).

  absorption and fluorescence emission spectra were red-shifted (λ abs = 529 nm, λ em = 542 nm) in comparison with 72 and 76 due to the additional ethyl groups in the α-position of the BODIPY fluorophore rendering it more electron rich (figure 63, left top and bottom). Besides the red-shifted absorption and emission, properties are reminiscent of the hydrosoluble BAPTA-BODIPYs with a Stokes shift of 460 cm -1 and large extinction coefficient (ɛ max = 56800). The maximum change in the fluorescence emission is obtained at 542 nm and 82 displays a low fluorescence quantum yield in the absence of Ca 2+ (ɸ em = 0.001), which is comparable to BAPTA-BODIPY (72) and BAPTA-PH-BODIPY (76) in absence of the ion.

Figure 63 :

 63 Figure 63: Fluorescence emission spectra of amphiphilic BAPTA-BODIPY (82) upon Ca 2+ -addition in pseudo intracellular buffer (λ exc = 496 nm) (top left). Hill plot for the spectral change in fluorescence emission at 542 nm (top right). Electronic absorption spectra of 82 upon Ca 2+ -addition (bottom left). Hill plot for the spectral change in electronic absorption at 265 nm (bottom right).

Figure 64 :

 64 Figure 64: Fluorescence emission spectra of C 8 F 17 -fura red (97) upon Ca 2+ -addition in pseudo intracellular buffer (λ exc = 496 nm) (top left). Hill plot for the spectral change in fluorescence emission at 680 nm (top right).Electronic absorption spectra of 97 upon Ca 2+ -addition (bottom left). Hill plot for the spectral change in electronic absorption at 496 nm (bottom right).

Figure 65 :Scheme 17 :

 6517 Figure 65 : Structural formulas of photolabile EDTA, EGTA and BAPTA-based Ca 2+ -cages and their respective photoproducts resulting in a decrease in binding affinity and a release of Ca 2+ -ions.

37

 37

  photoconversion to the 4-amino-2′-nitrosobenzophenone which has an electron withdrawing effect on the BAPTA unit. The change in binding affinity was measured to be 3000-fold compared to 40-fold in the presence of only one photodecaging group. The incorporation of two photolabile groups simultaneously increased the Ca 2+ decaging tremendously but EGTA based decaging agents still give higher affinity changes. The advantage is though that a relatively high release rate is combined with the pH insensitivity of BAPTA based Ca 2+binders.

Figure 66 :

 66 Figure 66: Recent developments in the Ca 2+ -decaging field. Left: nitr-T (102) a photolabile decaging agent with a binding affinity change of 3000-fold. Right: A redox triggered Ca 2+ -decaging agent based on ferrocene (103).

the 8 Scheme 18 :Scheme 19 :

 81819 Scheme 18: Synthetic route for the water soluble photo-labile Ca 2+ decaging agent DMNPE-4 (99).

Figure 67 :

 67 Figure 67: Spectral changes in the electronic absorption spectrum of 99 upon irradiation at 365 nm for 0, 7, 15, 20, 25 and 45 min using a mercury lamp with monochromator (see experimental section).

  (left) the fluorescence emission spectra of a solution containing 0.80 mM of DMNPE-4 (99) and 6 µM of BAPTA-BODIPY (72) in the presence of varying amounts of CaCl 2 is shown. The data can then be linearised using a Scatchard-type analysis shown in figure 68 (right).

Figure 68 :

 68 Figure 68: Fluorescence emission spectra of a solution containing 0.80 mM of DMNPE-4 (99) and 6 µM of BAPTA-BODIPY (72) in the presence of varying amounts of Ca 2+ in pseudo intracellular buffer (λ exc = 476 nm) (left). Scatchard plot of the Ca 2+ -titration (right).

Figure 70 :

 70 figure70. The same titration was performed using 99 after photolysis at 365 nm. Adding 1 to 10 equivalents of this photolysed solution to the same BAPTA-BODIPY (72) containing buffer yielded the curve shown in red dots in figure70.

Figure 71 :

 71 Figure 71: Fluorescence intensity at 512 (black), DMNPE-4 (99) and upon irradiation at 365 irradiation bench with 15 cm distance between sample and lamp.

)

  photoproducts of Ca 2+ -decaging agent(99) and the induced photobleaching of the fluorophore. The ion-sensor itself absorbs at 365 nm and the rather long irradiation time could explain the lowered maximum fluorescence emission observed.

  which have been concluded earlier, three novel amphiphilic Ca 2+ -ion sensors have been synthesized and show efficient binding under pseudointracellular conditions of pH and ionic strength. Ca 2+ -dissociation constants of 0.185 µM for BANIF (79), 0.724 µM for C 8 F 17 -BAPTA-BODIPY (82) and 0.076 µM for C 8 F 17 -fura red (97) had been determined via fluorescence titrations. The obtained values are in good agreement with known Ca 2+ -sensors in the literature and commercially available. The fluorescence enhancement of BANIF (79) (4.3-fold) and C 8 F 17 -BAPTA-BODIPY (82) (4fold) is somewhat diminished due to their amphiphilic nature and the aggregation of the molecules in aqueous media. C 8 F 17 -fura red (97) shows ratiometric behaviour in the absorption spectrum upon Ca 2+ binding which can be exploited for ratiometric measurements of Ca 2+ concentration in solution. In addition water soluble (99) and amphiphilic Ca 2+decaging agents bearing a hydrocarbon (122) and a fluorocarbon chain (124) have been synthesized which show a high affinity towards Ca 2+ (0.057 µM and 0.032 µM respectively). Upon irradiation and photocleavage of the Ca 2+ -binding site the binding affinity of these molecules decreases. Finally the Ca 2+ -based ion transfer between DMNPE-4 (99) and BAPTA-BODIPY (72) has been established under pseudo-intracellular conditions determining the molecular ratio of ion-ejector and ion-sensor for the following experiments in self-assembled systems. 4 Ca 2+ -transfer between self-assembled synthetic vesicles and in microcompartments 4.1 Introduction A wide range of techniques and molecules are known to form spontaneous organized supramolecular architectures such as micelles and vesicles. In this thesis the focus lies on amphiphilic structures consisting of molecules bearing a polar head group and a hydrophobic tail. These amphiphiles can self-assemble spontaneously into different structures depending on concentration, environment and temperature. Since the goal of the project is the formation of multicompartmentalized systems and the ion transfer between nano-objects inside, only persistent vesicle-forming amphiphiles are discussed in detail. 4.1.1 Amphiphiles and liposome formation Amphiphilic molecules dissolve in water at very low concentration but above a certain, amphiphile-specific concentration (the CMC or critical micellular concentration) they selfassemble in order to minimise unfavourable hydrophobic chain -water interactions and are held together by hydrophobic interactions. The type of assembly formed by amphiphiles depends partly on the molecular structure of the amphiphile or the surfactant packing parameter (S) which is defined by equation 20,

Figure 72 :

 72 Figure 72: Possible self-assembled structures depended on the

H 2 Figure 73 :

 273 Figure 73: Schematic representation of a phospholipid liposome in aqueous media.

  1964 by Bangham and Horne byThe resulting liposomes consisted of a lipid bilayer with a n aqueous core. The most popular example for liposomes are the ones consisting of a bilayer made of glycerophospholipid shown in figure Ever since their discovery, liposomes have been studied extensively for their properties biological membranes which consist largely of phospholipids (see The unique property of these capsules is the confinement of water-soluble molecules in their aqueous interior or incorporation of hydrophobic molecules in the lipid which resulted in many applications as drug delivery agents or for example as micro-Even though they are part of the cell membrane, phospholipids are not the only Schematic representation of a phospholipid liposome in aqueous media. dodecylammonium bromide (DDAB, sicles with a bilayer thickness From there on it became clear that a wide range of synthetic amphiphiles posses the ability to form bilayers or liposome-like structures, see examples in figure74.These amphiphiles can be cationic like DDAB (126), di-n-octadecylammonium bromide (DOAB, 127) and gemini amphiphiles with two ammonium headgroups or anionic like di-ndodecyl phosphate (DDP, 129) and di-n-hexadecyl phosphate (DHP, 130). Uncharged amphiphiles like polyglycerol (PGE, 128) or zwitterionic species like the phospholipid 1,2dimyristoyl-sn-glycero-3-phosphocholine (DMPC, 131) belong to this family as well.

Figure 74 :

 74 Figure 74: Molecular structures of some vesicle-forming synthetic amphiphiles.

10

 10

4. 1 . 2

 12 Fluorinated amphiphiles and vesiclesFluorinated amphiphiles have several advantages over their hydrocarbon analogues when it comes to vesicle formation. These amphiphiles consist of a polar headgroup, such as phosphocholine or ammonium, and a hydrophobic fluorocarbon tail. A schematic representation is given in figure75. The formation of vesicles by amphiphiles is based on the hydrophobic interaction of the hydrocarbon tail as mentioned earlier. In fluorocarbon

Figure 75

 75 Figure 75 Schematic representation of fluorinated amphiphiles and fluorinated

  figure 76.

Figure 76 :

 76 Figure 76: Schematic representation of a vesicle formed by a block copolymer (polymersome).

120 prepared and shown

  to form vesicles under certain conditions.20 A schematic representation for the structure of a classical polymersome consisting of a diblock copolymer is given in Schematic representation of a vesicle formed by a block copolymer (polymersome).olymersomes, like liposomes years before, have gained considerable interest in drug delivery applications due to their ability to encapsulate different molecules like drugs, DNA or siRNA in their aqueous core or hydrophobic molecules in their Additionally their circulation time in the human body is increased compared to liposomes and an increased stability results from a thicker membrane (3 though a lot of different methods for the preparation of polymeric vesicles are known a resemble those of liposomes, they follow mostly two main routes. The first is analogous to liposome formation via the hydration of a dried thin film with aqueous buffer yielding a polydisperse solution of different vesicles sizes. The second is the solvent switching method

Figure 77 :

 77 Figure 77: Schematic representation of the first preparation of a multicompartmentalized liposomes in a liposome (vesosome).

8 Figure 78 :

 878 Figure 78: Fluorescence emission at 512 nm (λ exc = 475 nm) of a solution containing formulation A, 5 µM of BAPTA-BODIPY (72) and CaCl 2 respectively upon irradiation at 365 nm using a classical TLC lamp.

Figure 79 :

 79 Figure 79: optical microscope image of formulation B (left) x 40 magnification. Epifluorescence image of the same region (λ exc = 330 -380 nm)

Figure 80 : 16 4. 2 . 2 . 1 Scheme 20 :

 801622120 Figure 80: Fluorescence emission at 512 BAPTA-BODIPY (72) and CaCl 2 respectively upon irradiation at 365

Figure 81 :

 81 Figure 81: Fluorescence emission of formulation C (λ exc = 425 nm) upon Ca 2+ -addition (left). Hill plot for the spectral change in fluorescence emission at 530 nm (right).

Figure 82 :

 82 Figure 82: Fluorescence emission of formulation D (λ exc = 485 nm) upon Ca 2+ -addition (left). Hill plot for the spectral change in fluorescence emission at 545 nm (right).

Kd = 3

 3

Figure 83 :

 83 Figure 83: Electronic absorption spectra of formulation E upon Ca 2+ -addition (left). Hill plot for the spectral change in the absorption spectra at 476 nm (right).

  As a representative example a formulation of BANIF(79) in fluorinated gemini (138) has been prepared. Because of the following experiments and the preparation of multicompartmentalized systems a concentration of 130 mg/ml of sucrose needed to be present in the buffer. Therefore formulation F contained 5 µM BANIF(79) in the membrane of 1.5 mM fluorinated gemini (138) vesicles in a pseudo-intracellular sucrose buffer (100 mM KCl, 30 mM MOPS, 130 mg/ml sucrose, pH 7.2). The fluorescence emission of this solution was then measured at 526 nm upon the addition of water-soluble DMNPE-4 (99) and subsequent irradiation at 365 nm. The resulting curve is plotted in Figure84.

Figure 84 :

 84 Figure 84: Fluorescence emission of formulation F at 526 (black). Fluorescence emission of the same solution after 0 to 6

Figure 84 :

 84 Figure 84: Schematic representation of the ion transfer between formulation G and F (top). Fluorescence emission of a 1:1 mixture of formulation F and G in the presence of 5increasing irradiation times at 365 nm using a TLC lamp.

  Schematic representation of the ion transfer between formulation G and F (top). Fluorescencelation F and G in the presence of 5 µM CaCl 2 at 526 nm (λ increasing irradiation times at 365 nm using a TLC lamp.

124 79 )132

 79 incorporated in the membrane of the vesicles with an amphiphile intracellular sucrose buffer has been prepared nd formulation G were mixed in equal volume ratio and was added, then the fluorescence emission of formulation F was recorded at nm. The obtained graph is shown in Figure 84. The fluorescence intensity emitted from e beginning of the experiment due to the presence of formulation G, nm the fluorescence increases s. A large jump in fluorescence intensity is observed between 60 and s of irradiation. The reason for the slow increase in fluorescence is the excess of ) present in the membrane of the vesicles in formulation G. Upon irradiation part of the photolabile decaging degrades and releases the Ca 2+ -ion, which is Schematic representation of the ion transfer between formulation G and F (top). Fluorescence nm (λ exc = 425 nm) after 138 After a sufficient amount of decaging agents are photolysed, a large increase in fluorescence intensity is noted due to the now more effective ion transfer from the ejecting vesicles (formulation G) to the accepting vesicles (formulation F). Above 200 s irradiation a slight decrease in fluorescence emission is observed due to photobleaching or other photodecomposition processes since the amphiphilic Ca 2+ -sensor in the membrane of the vesicles in formulation F also absorbs at 365 nm).In summary, the effective ion transfer from one vesicle formulation to the other has been shown and the ion transfer can be detected by means of fluorescence spectroscopy. This leads now to some first tests of multicompartmentalization.

Figure 85 :

 85 Figure 85: Multicompartmentalization of synthetic amphiphile polymersomes.

Figure 86 :

 86 Figure 86: Schematic representation of the molecular composition of formulation H containing C 8 F 17 -BODIPY (150) and formulation I bearing dodecyl fluorescein in the bilayer.

  microscopy. Bright field and fluorescence images of the emulsion of formulation H in water droplets stabilized by PB-b-PEO are depicted in figure 87.

Figure 87 :

 87 Figure 87: Bright field (left), fluorescence (middle) and overlay of bright field and fluorescence image (right) of a formulation H in a water in oil emulsion via PB-b-PEO.

Figure 88 :

 88 Figure 88: Bright field (left), fluorescence (middle) and overlay of bright field and fluorescence image (right) of a formulation H in the aqueous interior of a PB-b-PEO polymersome.

Figure 89 :

 89 Figure 89: Fluorescence image of formulation I (left), Bright field (middle) and fluorescence image (right) (λ exc = 476 nm) of a formulation I after the emulsion centrifugation procedure with PB-b-PEO.

Figure 89 :

 89 Figure 89: Schematic representation of the molecular composition of formulation J containing C (152) in the bilayer.

  it was assumed that two different formulations of fluorinated vesicles could coexist at the same time in the interior of the and therefore another formulation bearing a green emitting fluorescein based ) in the membrane has been prepared. The synthesis of this dye is shown in chapter 5. A schematic representation of the molecular composition of formulation J is shown in Schematic representation of the molecular composition of formulation J containing C µM C 8 F 17 -fluorescein (150) in the bilayer of fluorinated vesicles phile concentration of 1.5 mM in pseudo-intracellular sucrose buffer was nm membrane to obtain vesicles with an average diameter of 396 Formulation J and formulation H were then mixed in equal volume ratios. The obtained was subsequently used in the emulsion-centrifugation procedure used for the previous results. In figure 90 confocal fluorescence microscope images are depicted of the obtained emulsion and the polymersomes after centrifugation. 152 138 hybrid vesicles. One characteristic of these types of structures are areas of higher and lower amphiphile content which can be seen in areas with no apparent fluorescence in the bilayer made previously in the literature for lipid Although this was a negative result in terms of coencapsulation of the two different types of vesicle formulations these structures have a certain interest for permeability which will not be further discussed here. it was assumed that two different formulations of fluorinated vesicles could coexist at the same time in the interior of the and therefore another formulation bearing a green emitting fluorescein based ) in the membrane has been prepared. The synthesis of this dye is shown in chapter 5. A schematic representation of the molecular composition of formulation J is shown in Schematic representation of the molecular composition of formulation J containing C 8 F 17 -fluorescein ) in the bilayer of fluorinated vesicles intracellular sucrose buffer was nm membrane to obtain vesicles with an average diameter of 396 nm. Formulation J and formulation H were then mixed in equal volume ratios. The obtained centrifugation procedure used for the previous results. In figure 90 confocal fluorescence microscope images are depicted of the obtained

Figure 90 :

 90 Figure 90: Bright field and fluorescence overlay images of the emulsion containing formulation H and J at the same time in the PB-b-PEO stabilized droplets (left). Bright field and fluorescence overlay image of the resulting polymersome after emulsion-centrifugation (right)

Figure 91 :

 91 Figure 91: Schematic representation of the system prepared for the ion transfer between water molecules in PB-b-PEO vesicles.

Figure 92 :

 92 Figure 92: Confocal fluorescence images of polymersomes containing Ca and 5 µM Ca2+-sensor (8) in the aqueous lumen of the polymersome.

Figure 93 :

 93 Figure 93: Average fluorescence emission of 12 independent polymersomes containing Ca (99) 5 µM CaCl 2 and 5 µM Ca 2+ -sensor ( at 488 nm and a short laser pulse at 405 between 510 and 650 nm.

  . The fluorescence intensity in the beginning to the decaging agent (99) and the nm is given. Average fluorescence emission of 12 independent polymersomes containing Ca 2+ -decaging agent ) in the aqueous interior of the polymersome. Continuous irradiation nm were applied and the fluorescence intensity recorded over time nm the fluorescence intensity increases sharply. The intensity. After the ions were transferred ) during the laser pulse, the fluorescence keeps decreasing slowly, which ). However this experiment is nm due to the use of a less strong ions can be released "on demand" at any given moment during the experiment. After it was shown that the ion transfer in confined is indeed possible the ion transfer between vesicles in the aqueous interior of the 4.4.1 Ca 2+ sensing vesicles in the confocal microscope Three formulations of Ca 2+ -sensing vesicles have been prepared in order to find the most appropriate formulation for studies with the confocal microscope. Formulation K consisted of 5 vesicles were prepared via thin film hydration using a pseudo 10 mM EGTA. The sample was extruded through a 800 multilamellar structures. After the preparation the sample was split in two and the CaCl added to one part to obtain a free Ca of formulation K and the fluorescence emission spectra in solution and the fluorescence emission obtained via confocal microscopy were compared. While the formulation K in the presence of Ca 2+ is clearly more fluorescent regarding the emission spectra measured spectrofluorometer in this case is more complicated while observing the sample via confocal microscopy (figure 94, bottom right). Here the fluorescence emission shows the expected difference in fluorescence emission only in the beginning of the experi

Figure 94 :

 94 Figure 94: Schematic representation of formulation K (left). Fluorescence emission spectra of formulation K in presence and absence of Ca 2+ (λ em measured via confocal microscopy (λ

Figure 95 :

 95 Figure 95: Schematic representation of formulation L (left). Fluorescence emission spectra of formulation L in presence and absence of Ca 2+ (λ em measured via confocal fluorescence microscopy (λ

Figure 96 :

 96 Figure 96: Schematic representation of formulation M (left). Fluorescence emission spectra of formulation M in presence and absence of Ca 2+ (λ em measured via confocal microscopy (λ

  contrary to vesicle formulation K the fluorescence intensity stabilizes itself well below the fluorescence emission of formulation therefore a suitable candidate for the studies via The incident increase in fluorescence emission might be explained by low levels of impurities in the preparation of formulation L or a partial toreaction would probably consume all molecules and can therefore be excluded. The last formulation which was studied was formulation M, which was furared (97) in the bilayer intracellular sucrose-containing Schematic representation of formulation M (left). Fluorescence emission spectra of formulation M in nm) (top right). Fluorescence emission between 510 and 810 nm Molecular composition of formulation M, the emission spectra observed via a over time measured under a confocal fluorescence microscope are shown in figure 96. The fluorescence emission spectra also show a clear difference in the emission intensity in presence and absence of Ca 2+ . It is noteworthy is lower than in the absence,

4. 4 . 2

 42 Ion transfer between fluorocarbon vesicles in PB-b-PEO polymersomesIn order to test the ion transfer with Ca 2+ -sensing vesicles in confined polymer compartment, a formulation of C 8 F 17 -BAPTA-BODIPY (82) bearing vesicles has been prepared (formulation N). The concentration of C 8 F 17 -BAPTA-BODIPY (82) was 5 µM and the fluorinated amphiphile 1.5 mM in pseudo-intracellular sucrose-containing buffer. The formulation was extruded through a 800 nm membrane to omit large multilamellar structures.Water soluble DMNPE-4 (99) was added until a final concentration of 75 µM of the Ca 2+decaging agent was reached and subsequently 5 µM of CaCl 2 . This mixture was then subjected to the emulsion-centrifugation procedure using PB-b-PEO copolymer to obtain polymersomes with formulation N and DMNPE-4 (99) in the aqueous interior. A schematic representation of the system is given in figure97. The fluorescence of these polymersomes was been recorded via confocal fluorescence microscopy. The fluorescence emission of 10 independent polymersomes with approximately the same size has been observed between 510 -650 nm upon exciting the system at 488 nm. After 420 ms a laser pulse of 40 ms with maximum intensity at 405 nm was sent in order to photo-decage the bound Ca 2+ . The observation made is plotted in figure 98 (black).

Figure 97 :

 97 Figure 97: Schematic representation of the ion transfer between water-soluble DMNPE-4 ( bilayer of the fluorinated vesicles.

Figure 98 :

 98 Figure 98: Fluorescence intensities over time (left) for a solution containing formulation N, 75 (42) and 5 µM CaCl 2 (λ exc = 488 nm). A short laser pulse (40 405 nm (black) or at 488 nm (red). Confocal fluorescence image of a formulation N containing PB polymersome (right).

Figure 99 :

 99 Figure 99: Schematic representation of the prepared PB formulation N simultaneously in the aqueous compartment. The light interior of the polymersome and the molecular composition is shown on

Figure 100 :

 100 figure 100. The previously described FRAP experiment was used to observe the transfer partment. Briefly, after nm was applied (FWHM = from the ejector vesicles (formulation O), while the fluorescence nm. The observed fluorescence was normalized as shown before and the average fluorescence of 10 polymersomes has been taken to obtain the curves depicted in figure 100. The 1:1 mixture of formulation N and O yielded anm is in fact smaller than the initial intensity. This is due to ratio between ion transferred and photobleaching, which is unfavorable in this case. However, the fluorescence intensity increases slightly after the laser

  Left: Fluorescence intensities as a function of time recorded via confocal fluorescence microscopy is applied. Mixtures of formulation O and N (3:1, black and 1:1 red) were studied and compared to a 1:1 mixture of formulation O and nm. Right: confocal fluorescence microscopy image of a characteristic The 3:1 mixture of formulation O and N gives a different picture. The fluorescence intensity nm is elevated by a factor of 1.2 as previously observed for the ) and formulation N. The final mM fluorinated gemini (138) mM fluorinated gemini ) in formulation N. With this ratio between sensing vesicles the ion transfer seems to work efficiently. It rather difficult since it impurities in the glassware, the purity of the and the ion impurities in the reagents used.

Figure 101 :

 101 Figure 101: Fluorescence intensities between 510 and 650 fluorescence microscopy of 10 polymersomes (λ pulse at 405 nm (blue bare) to release Ca at 488 nm (blue bare). The fitting of the first 2.4

  405 nm light pulse at 488 nm Fluorescence intensities between 510 and 650 nm as a function of time recorded via confocal fluorescence microscopy of 10 polymersomes (λ exc = 488 nm) of a mixture of formulation O and N (3:1). A laser nm (blue bare) to release Ca 2+ is applied (black) and compared to the same sample with a laser pulse nm (blue bare). The fitting of the first 2.4 s of the experiment is shown (inlet). normalized average fluorescence intensities of 10 polymersomes over time are plotted in figure 101. The ion transfer seems to work effectively also with the shortened laser pulse as can be seen in the rise of fluorescence after the laser pulse at 405 nm where formulation O does not absorb light is unaffected (red). The slow photobleaching process after the laser pulse and continuous irradiation at 488 are similar to previous experiments. Mono-exponential fitting of the first 2.4 the laser pulse and extrapolating the 20 ms during the pulse to the initial intensity, gave a rate . This corresponds in the order of magnitude to the slow Ca component measured in the group of Ellis-Daviess for DMNPE-4 derivative (1.5

  process. For this experiment the same ratio between formulation O and formulation N was used as in the previous experiment but the laser pulse was kept as short as possible (20 ms) in nm as a function of time recorded via confocal of formulation O and N (3:1). A laser is applied (black) and compared to the same sample with a laser pulse normalized average fluorescence intensities of 10 polymersomes over time are plotted in figure101. The ion transfer seems to work effectively also with the shortened laser pulse as can be seen in the rise of fluorescence after the laser pulse at 405 nm (black). A nm where formulation O does not absorb light is unaffected (red).

  be divided into three different types namely ρ, different sensitivity maxima at 590 nm (orange colour matching functions (CMF) representing their spectral sens two independent experiments conducted by Wright recognised by the human, could be expressed through a mathematical formalism. These CMF showed some negative values and were therefore transform values appear. The standard observer based on these experiments was then defined by the Commission Internationale de l'Éclairage colour matching functions ̅ݔ (λ) , 102.

Figure 102 :

 102 Figure 102: Colour matching functions for the standard observer according to CIE1931.

Figure 103 : 22 equation 23 The

 1032223 Figure 103: The x-y plane of the CIE 1931 with wavelength (nm) are indicated.

5. 1 . 1

 11 White light emission in supramolecular chemistryWhite light emission in supramolecular assemblies has gained considerable interest in the last few years due to their use in lighting and displays4 . Many different approaches had been shown towards white light emission in these systems, most of the time this is achieved by combination of red, green and blue emitting dyes. When different chromophores are located close in space as one may anticipate in a discrete supramolecular assembly, Förster energy transfer often occurs between donor and acceptor fluorophores, which participates in defining the output colour and thus in obtaining white light.

Figure 104 :

 104 Figure 104: Schematic representation of the spectral overlap of fluorescence emission of a donor and the absorption spectrum of an acceptor.

acceptor can be calculated according to equation 26 ܬሺߣሻ≡

 26  ɛ ሺߣሻߣܨ ሺߣሻ݀ߣ ∞ stands for the extinction coefficient of the acceptor in M normalized intensity of the donor emission spectrum and dλ is the wavelength range of the Schematic representation of the spectral overlap of fluorescence emission of a donor and the ling these formulas the distance between two fluorophores can be calculated by measuring the Förster energy transfer. Vice versa knowing the distance between two fluorophores the efficiency of the energy transfer can be calculated. white light emitting polymers and polymer films has been presented

Figure 105 :

 105 Figure 105: Molecular structure and schematic representation of the formation of white light emitting organogels 8 .

Figure 106 :

 106 Figure 106: Preparation process of white light emitting ink using a liquid OPV and blending it with green emitting Alq3 and red emitting rubrene.

)

  introduced by Yang et.al. emits white light only under very specific conditions like certain pH, solvent and irradiation wavelength. On the other hand with this fluorophore the emitted spectra can be tuned changing the named parameters. In the group of de Cola a white light emitting europium/indium complex (147) has been synthesized. Due to energy transfer with an efficiency of 38 % from the indium complex to the europium, center ission has been created.Preparation process of white light emitting ink using a liquid OPV and blending it with green While the previous examples made use of energy transfer between fluorophores to achieve white light emission another approach is the usage of a single white light emitting fluorophore. An example of the three different white light emitting dyes is given in figure107. These molecules have the emitting dyad (145) creates the white light spectrum by frustrated energy transfer due to excited state raphenyl imidazole unit to the other. The emits white light only under very specific conditions like certain pH, solvent and irradiation wavelength. On the other hand with this can be tuned changing the named parameters. In the group of ) has been synthesized. Due to % from the indium complex to the europium, center

Figure 107 :

 107 Figure 107: White light emitting dyad by Park, S. et. al. (145) and white light emitting benzo[a]xanthene by Yang, Y. et. al. (146) and white light emitting europium / iridium complex (147) by Coppo et. al.

Figure 108 :

 108 Figure 108: White light emitting spin coated film of red and green emitting polymer micelles and blue emitting polyfluorene in the periphery.

5. 2

 2 Synthesis of RGB fluorophores based on coumarin, fluorescein and BODIPY The red light emitting fluorophore was based on the design of the perfluorinated BODIPY synthon which features a perfluorinated phenyl ring described in chapter 2 of this thesis similar fluorophore has been published by Galangau 162 light emitting spin coated film of red and green emitting polymer micelles and blue emitting

Scheme 22 :

 22 Scheme 22: Synthesis pathway of green light emitting C 8 F 17 -fluorescein (152).

23 Scheme 23 :

 2323 Scheme 23: Synthesis pathway of blue light emitting C 8 F 17 -pacif (156).

Figure 109 :

 109 Figure 109: Graphical representation of the particle size measured by DLS for Formulation R1 (red), G1 (green) and B1 (blue).

  of 10 µM of fluorophore in the membrane of the vesicles. However, due to the large aromatic nature of the molecular structure the incorporation into membrane seemed unefficient and only 3.2 µM of fluorophore were loaded. During the extrusion of the vesicles formulation a big part of the compound was precipitated on the extrusion membrane. Incorporation of Pacific-C 8 F 17 and Fluorescein-C 8 F 17 proceeded more smoothly aiming for a concentration of fluorophore in the membrane of 5 µM (4.2 µM incorporated) and 20 µM (20.3 µM incorporated), respectively. 5.4 Spectral characterisation of fluorophores BODred (150), C 8 F 17fluorescein (152), Pacific-C 8 F 17 (156) and vesicle formulation B1, G1 and R1 Electronic absorption and emission spectra of the fluorophores were recorded in solution and incorporated in the vesicle membrane. The spectra are depicted in figure 110. The absorption spectrum of the pacific-C 8 F 17 (156) shows a small hypsochromic shift of 3 nm upon incorporation into the membrane which can be attributed to the more apolar environment in the vesicle membrane. However, fluorescein-C 8 F 17 (152) shows a small batochromic shift of 5 nm in the vesicle membrane. To explain this behaviour two effects need to be considered. First, the effect of a more hydrophobic environment in the membrane leading to a blue-shift and second the physical separation of the fluorophores from each other. Molecules carrying a fluorinated chain tend to aggregate much easier due to their increased lipophilicity compared to their hydrocarbon analogs. The observed change in the absorption spectra is therefore a combination of the two effects. BODred (150) shows a small hypsochromic shift of 6 nm and behaves therefore more like the parent pacific blue derivative. For all three fluorophores a broadening of the absorption band has been observed due to the change in rotational and vibrational states in the amphiphile membrane.

Figure 110 : 27 For

 11027 Figure 110: Absorption (left) and emission spectra (right) of pacific-C 8 F 17 (156) (MeOH, λ exc = 405 nm), fluorescein-C 8 F 17 (152) (MeOH, λ exc = 458 nm), BODred (150) (THF, λ exc = 458 nm) and Formulation R1, G1 and B1 in pseudo intracellular sucrose buffer( λ exc = as corresponding fluorophore).

  is increased by 8% compared to the molecule in solution. Taking the error of the measurement into account the values measured are comparable. The BODIPY derivative (150) however shows a decrease of 35 % on average when incorporated in the membrane of the vesicles.Since the emission of BODIPY meso-subustituted with phenyl groups depend on the planarity of the structure and considering the size of the red light emitting elongated BODIPY, one can reason that there might a certain steric effect in the vesicle membrane and therefore a deformation of the BODIPY core and a loss of fluorescence emission. The influence of the orientation of BODIPY in a polymer film on the fluorescence quantum yield has been studied24 . It was shown that depending on the polymer used (examples are given for different methacrylates like PMMA or fluorinated polymethacrylates like PFMA) and the orientation of BODIPY in the polymer film can lead to a 20 % lower quantum yield.5.5 White light emission of mixed fluorocarbon vesicles bearing red, green and blue fluorophoresIn order to obtain a white light emitting solution of vesicles one needs to determine the ideal ratio between red, green and blue light emitting formulations. To obtain the approximate ratio, titrations between blue and green blue and red emitting vesicle formulations had been conducted. Formulation B1 which has the coordinates (x = 0.1514 / y = 0.0880) is the most outer point in the blue part of the CIE1931 graph represented in figure11. Formulation G1 with the coordinates (x = 0.3271 / y = 0.6322) is the most outer point in the green region of the graph and Formulation R (x = 0.6719 / y = 0.3012) is the outer point in the red region.

Figure 111 :

 111 Figure 111: left: CIE1931 colour space of formulation R1 G1 B1. D65 white standard and coordinates for formulation W are highlighted (λ exc corresponding locations in the colour space. Right: W.

  formulation W (x = 0.30639 / y = 0.32164) are depicted in figure 111 and are in close proximity. On the right side of the same figure the confocal microscope image is shown. Using a confocal microscope, the macroscopically observed white light e down to its blue green and red parts and single separate vesicles can be observed, where each outer point in the blue part of the CIE1931 graph represented in figure 11. Formulation G1 with the coordinates (x = 0.3271 / y = 0.6322) is the most outer point in the green region of the graph and Formulation R (x = 0.6719 / y = 0.3012) is the outer point in the red region. Mixtures of two formulations had been prepared in the ratio 1/4, 2/3. 1/1, 3/2 and 4/1 (v/v). between the coordinates left: CIE1931 colour space of formulation R1 G1 B1. D65 white standard and coordinates for O). Images of the different formulations are placed beside their = 405 nm) of formulationThis is a general behaviour of mixed chromophores since the resulting colours of the mixed system are always a combination of the two emission spectra. The coordinates for the D65 ard were envisaged for the white light emitting vesicle formulation. An appropriate mixture of formulation B and G was taken (1 eq. form. B1 / 2 eq. form. G1) and doped with formulation R to obtain a white light emitting mixture which will be called eq form. G1/ 2.5 eq ), 7.38 µM (152) and ). The coordinates for the white light standard (x = 0.31271 / y = 0.32902) and formulation W (x = 0.30639 / y = 0.32164) are depicted in figure111and are in close proximity. On the right side of the same figure the confocal microscope image is shown.

Figure 112 :

 112 Figure 112: Fluorescence timetraces observed in a 1 femtoliter volume of formulation W (λ exc = 375 nm). Left:blue emission (375 -475 nm) and green emission (500 -580 nm), middle: blue emission (375 -475 nm) and red emission (570 -720 nm), right: green emission (500 -580 nm) and red emission (570 -720 nm).

Figure 113 :

 113 Figure 113: Spectral overlap for the emission of formulation B1 and the absorption of formulation G1 (left).Spectral overlap for the emission of formulation G1 and the absorption of formulation R1 (right).

10 16 M

 16 -1 cm -1 nm4 which yields a Förster radius (R 172 incorporated in the membrane with a shorter lifetime component of 2.23 ting that most of the fluorophore is located in the core region. C ) has a lifetime which is shortened (from 4.88 ns to 4.12 indicating a closer proximity to the polar head groups with a shorter lifetime component % of the total lifetime. This behaviour can be explained by the increased size of the molecule, compared to fluorophore 152. Molecule BODred ( behaves similarly to molecule 152 showing a shorter lifetime in the membrane (from 5. ns in vesicles) but with a higher percentage of the shorter lifetime component %). Again the elongated BODIPY structure is larger than the fluorescein core and therefore penetrates deeper into the hydrophilic part of the membrane. The longer lifetime components in formulation W have approximately the same values as in formulation R1, G1

43 10 which

 10 results in an estimated Förster radius (R 0 ) of 8.4 nm calculated by equation J value for the green-red overlap has been calculated to be 18.1 which yields a Förster radius (R 0 ) of 9.8 nm. One can reason that incorporated in the membrane with a shorter lifetime component of 2.23 ns with 14 % ting that most of the fluorophore is located in the core region. C 8 F 17ns to 4.12 ns) in membrane indicating a closer proximity to the polar head groups with a shorter lifetime component % of the total lifetime. This behaviour can be explained by the . MoleculeBODred (150) showing a shorter lifetime in the membrane (from 5.28 ns ns in vesicles) but with a higher percentage of the shorter lifetime component %). Again the elongated BODIPY structure is larger than the fluorescein core and embrane. The longer lifetime components in formulation W have approximately the same values as in formulation R1, G1 and B1, which implies that no energy transfer between the vesicles occurs and the Förster energy transfer would be anticipated due to spectral overlap and the fluorescence lifetime of the blue and green emission would be shortened. Figure113shows the spectral overlap of the absorption and Spectral overlap for the emission of formulation B1 and the absorption of formulation G1 (left).Spectral overlap for the emission of formulation G1 and the absorption of formulation R1 (right). transfer are given. The spectral overlap value has been calculated using equation 26. The value for the blue green overlap is 3.43 10 16 M - nm calculated by equation red overlap has been calculated to be 18.1 nm. One can reason that fluorophores incorporated in the vesicle membrane never come close enough to each other to undergo energy transfer. In addition being incorporated in the membrane the rotational freedom is reduced which results in an increased difficulty to align the dipoles and therefore К will be reduced.5.6 White light emissionGiant white light emitting polymersomes had been prepared by emulsion centrifugation using formulation W as described in the experimental section and in chapter 4. A sample of formulation W containing 380 polybutadien-polyethylenoxide block copolymer in a solution of toluene. This emulsified solution was placed on top of an aqueous solution containing 380 same ionic strength as formulation W. The remaining block copolymer forms a monolayer at the interface and after centrifugaten, emulsified vesicle containing droplets will pass through the monolayer into the aqueous layer to form multicompartmentalized polymersomes according to the procedure published by Marguet temperature for at least one hour to obtain giant polymersomes in solution. A schematic representation of the preparation process is shown in figure114.

Figure 114 :

 114 Figure 114: Schematic representation for the encapsulation process of formulation W and the p white light emitting polymersomes.

Figure 115 :

 115 Figure 115: Confocal microscope image of a giant polymersomes. Red (600 and blue (410 -490 nm) detector channels are shown separately and the overlay shows the three same time (λ exc = 405 nm; pinhole = 100

Figure 116 :

 116 Figure 116: Confocal microscope image of a giant polymersome using an increased pinhole value (300 Blue, green and red detector channels on left and the ove sensitivity, on the right (λ exc = 405 nm).

Figure 117 :

 117 Figure 117: Fluorescence emission spectra of formulation W and fluorescence emission spectra of white light emitting polymersomes recorded with a confocal microscope coupled to a spectrometer.

  spectra was measured by a fluorescence spectrometer coupled to the confocal microscope. An overlay of the emission spectra of formulation W and the spectra measured inside the polymersome is depicted in figure 117. In the fluorescence spectrum of formulation W (red line) all three emissions corresponding to formulation R, G and B can be attributed without any deformation of the emission maxima. This is another indication for an absence of energy transfer between the fluorophores. The emission spectrum of the giant polymersome (black line) matches the emission spectrum of formulation W, with only minor intensity differences.These differences are related to the different set up and excitation power used for the measure a fluorescence spectrum with a confocal microscope the sample needs to be irradiated more strongly since a much smaller volume is observed and therefore much less light is emitted. Fluorescence emission spectra of formulation W (red) recorded on a steady-state spectrofluorometer and fluorescence emission spectra of white light emitting polymersomes recorded with a confocal microscope Using a high laser intensity bleaches the fluorophores faster and during the accumulation of measured spectra the participation of the least stable fluorophore decreases, which is in the current experiment is the pacific blue derivative 156. The green (152 Confocal microscope image of a giant polymersome using an increased pinhole value (300 nm). rlay of the three detectors, tuned to the same equal In order to prove that the white light emission observed in the giant polymersome is the same lation W, the emission spectra was measured by a fluorescence spectrometer coupled to the confocal microscope. An overlay of the emission spectra of formulation W and the spectra measured inside the e spectrum of formulation W (red line) all three emissions corresponding to formulation R, G and B can be attributed without any deformation of the emission maxima. This is another indication for an absence of energy emission spectrum of the giant polymersome (black line) matches the emission spectrum of formulation W, with only minor intensity differences. These differences are related to the different set up and excitation power used for the measure a fluorescence spectrum with a confocal microscope the sample needs to be irradiated more strongly since a much smaller volume is observed and state spectrofluorometer and fluorescence emission spectra of white light emitting polymersomes recorded with a confocal microscope ing the accumulation of measured spectra the participation of the least stable fluorophore decreases, which is in the 152) and red (150) fluorophores appears to resist the harsh irradiation conditions. Decreasing the blue intensity, the coordinates of the white light emission are green and red-shifted. The new coordinates determined by confocal microscopy are x = 0.3057 / y = 0.3483 (form. W x = 0.30639 / y = 0.32164) which is still in close proximity to the D65 white light standard and corresponds well to a white light emission. The CIE1931 colour space with all measured colours is shown in figure 117 on the right side. In figure 118 giant white light emitting polymersomes are shown in the presence of formulation W. The image has been recorded with increased pinhole value (300 nm) to obtain pseudo-epifluorescence. White light emitting polymersomes can be identified in the presence of red, green and blue vesicles. This image shows that the white light emission occurs due to confinement in the giant polymersome. Vesicles which are not confined can freely move on the cover slide and spread out over the surface, which decreases the local concentration of vesicles in the observed volume. In the interior of the polymersomes the local concentration is preserved and white light emission can occur.

Figure 118 :

 118 Figure 118: Confocal microscope image of a sample of white light emitting polymersomes in the presence of form. W. Observation with increased pinhole value (300 nm).

Fluorescent 30 ɛ

 30 water soluble BAPTA-BODIPYs (72 and 76) showed a fluorescence enhancement of 122 and 23 -fold respectively upon Ca 2+ complexation under pseudointracellular conditions. The rate of photoinduced electron transfer has been determined via transient absorption measurements to be 3.3 x 10 12 s -1 for BAPTA-BODIPY (72) and 8.3 x 10 11 s -1 for BAPTA-PH-BODIPY(76) with K d values of 0,53 and 0,16 µM respectively rendering these molecules interesting molecules for ion sensing in biological systems. Amphiphilic Ca 2+ -switches based on the BAPTA ionophore and BODIPY(82), naphthalimide(79) and furan(97) fluorophores have been designed. Ca 2+ complexation studies yield binding affinities in the range of 0.076 -0.724 µM but with rather low fluorescence enhancement (4.3-fold max.) upon ion binding, which was attributed to aggregation in aqueous media.Water soluble DMNPE-4 (99) and amphiphilic (122 and 124) Ca 2+ -decaging agents have been designed based and their decaging properties tested under pseudo-physiologiccal conditions K d values and decaging efficiencies resemble those determined for DMNPE-4. The amphiphilic hydrocarbon analogue (122) is a promising candidate for applications in biological studies where Ca 2+ from lipid membrane surfaces is desired. Equally, the amphiphilic decaging agents have been incorporated into the self-assembled membrane of synthetic amphiphiles and their decaging properties have been verified. The amphiphilic Ca 2+sensors(79,82 and 97) showed increased fluorescence emission in vesicle membranes (one order of magnitude for 79 and 97 and two orders of magnitude for 82). These amphiphilic fluorophores could be used for the sensing of Ca 2+ close to biological membranes when equipped with hydrocarbon instead of fluorocarbon chains. Also the observed effect, namely the decrease in binding affinity towards Ca 2+ ,which was observed in cationic vesicles (circa 100-times lower for 82 and 97 and 10-times lower for 79) would be probably present to a lesser extent in biological membranes since the charge on the surface is not exclusively positive.Nevertheless, photoinduced Ca 2+ transfer had been successfully shown between decaging vesicle-in-polymersome stuctures were achieved when using fluorocarbon vesicles, while hydrocarbon vesicles formed hybrid amphiphile polymer capsules which will be an interesting target for membrane permeability studies in the future, for example for oxygen transport etc. The ion transfer between fluorocarbon vesicles in the aqueous interior of polymersomes had been achieved using UV-light as release trigger. The increase in fluorescence intensity of ion sensing vesicles gave direct proof of the occurrence of ion transfer. Fitting of the transfer kinetics revealed a slowed down decaging kinetic for DMNPE-4 based Ca 2+ -decaging agents in aqueous polymer compartments. This multicompartmentalized system with Ca 2+ ions bound to one internal vesicle could be exploited for more sophisticated architectures in the future. One could imagine internalization of biomolecules which are activated by Ca 2+ incorporated in the membrane of one vesicle and the Ca 2+ -decaging agent to another. Upon photodecaging biochemical reactions in confined compartments could be photocontrolled biochemical mircroreactors. In the last part, the multicompartmentalization approach was extended to three different types of vesicles in the internal compartment of a giant polymersome. By incorporation of independent red (150), green (152) and blue (156) light-emitting dyes in the membrane of fluorinated vesicles, white light emission in the microcompartment of the polymersome could be achieved as a result of nanoconfinement. This system containing three types of 200 nanometer-sized vesicles in a micrometric polymersome could be exploited for cascade reactions in microenvironements.In summary the goal of the project, namely the photoinduced ion transfer between synthetic vesicles in confined polymer compartments has been achieved and artificial mimicry for cellular communication has been shown. The most performant multicomponent system, namely fluorinated vesicles in polymersomes, opens the way towards some sophisticated future applications. The fluorescence quantum yield was determined by comparison to an optically dilute fluorescence standard with a known quantum yield according to the Parker method. 1 Mainly three different standards have been used namely fluorescein in 0.1 M NaOH (ɸ = 0.95) for green emitting dyes, 9,10-diphenylanthracene in cyclohexane (ɸ = 0.90) for blue emitting dyes and rhodamine 101 in ethanol containing 0.01 HCl (ɸ = 1.00) for red emitting dyes. The fluorescence quantum yield of the sample solution (ɸ) has then been calculated by equation xx1 where ɸ R is the fluorescence quantum yield of the reference, I is the integral of the fluorescence emission of the sample solution, I R is the integral of the fluorescence emission of the reference, A is the absorption at the excitation wavelength and ɳ is the refractive index of the solvent used. Transient absorption spectroscopy allows the observation and measurement of the evolution of the excited state of a molecule directly after its excitation and species with a very short time constant can be observed such as radicals or radical anions as well as dark non-radiative intersystem crossing processes giving triplet states. Two set-ups have been used to obtain transient absorption data, a femtosecond set up (1 ns -200 fs) and a picosecond setup (1 ms -1 ns). Transient absorption maps were constructed as follows: In order to generate the pumpprobe pulses a tripled frequency Nd: YAG amplified laser (30 ps, 30 mJ, 1064 nm, 20 Hz, model Ekspla 401) was used to pump an optical parametric generator (PG model Ekspla 401) in order to produce the excitation pulse in the wavelength range between 410 -2300 nm. The residual laser beam is focalised in a cell under high pressure of Xe, where a white light pulse is produced to measure the sample. All signals are analysed by a spectrograph (Princeton Instruments Acton model SP2300) coupled to a camera (Hamamatsu C7700). The accumulation of the pulses is stored and treated with the program HPDTA (Hamamatsu) in order to produce the transient absorption maps in two dimensions (wavelengths vs. time delay) of the absorption intensities between 300 and 800 nm. concentration of the light induced change C i (isomerisation or photocleavage) can be calculated as follows: is the extinction coefficient of the starting material and ɛ product is the exctinction coefficient of the photoproduct. The photochemical quantum yield is then calculated as shown in equation xx6. is the irradiated volume of the sample and t stands for the irradiation time of the sample.Calcium titrationTwo Calcium calibration stock solutions have been prepared, one containing 10 mM EGTA (0 µM Ca 2+ -buffer) and another with 10 mM CaEGTA (39 µM Ca 2+ -buffer). In addition both buffers were charged with 100 mM KCl and 30 mM 3-(N-Morpholino)-propanesulfonic acid (MOPS) adjusted at pH 7.2. In both buffers an equivalent amount of Ca 2+ -binding probe was dissolved with a final concentration between 1-10 µM. In order to determine the K d both fluorescence and electronic absorption spectra have been taken after having adjusted the free Ca 2+ concentration to 0.017 µM, 0.038 µM, 0.065 µM, 0.100 µM, 0.150 µM, 0.225 µM, 0.35 µM, 1.35 µM and 39 µM. Therefore 2 mL of the 0 µM Ca 2+ -sample was placed in a cuvette and the spectra were recorded. 200 µL of the sample was discarded and replaced with 200 µL of the 39 µM Ca 2+ -sample. In that fashion a concentration of 0.017 µM Ca 2+ can be obtained without changing the concentration of the analyte. The aforementioned Ca 2+ concentrations were then adjusted by discarding 250, 222, 250, 286, 333, 400, 500, 667 and 1000 µL and replacing the removed volume with an equivalent volume of the 39 µM Ca 2+ sample. For the calculation of the free Ca 2+ -concentration a constant room temperature of 21 °C was assumed.

19 F

 19 NMR (400 MHz, CDCl 3 ): δ = -146.74 (q, 2F, J = 32.3 Hz), -147,60 (m, 2F), -161,61 (m, 2F). HRMS (ESI): Calc. m/z = 490.1859 for C 23 H 24 B N 3 F 6 Na Found m/z = 490.1860 [M+Na] +Et 2 N-BODIPY(62)

13 C

 13 NMR (75 MHz, CDCl 3 ): δ = 206.12, 123.40, 194.08, 155.78, 30.13, 26.85, 12.92, 12.72.19 F NMR (400 MHz, CDCl 3 ): δ = -143 (m, 2F), -147 (m, 2F), -153 (m, 2F). ).HRMS (ESI): Calc. m/z = 490.1859 for C 23 H 24 B N 3 F 6 Na Found m/z = 490.1854 [M+Na] +azide-BODIPY(63)

13 C

 13 NMR (75 MHz, CDCl 3 ): δ = 156.14, 136.80, 133.97, 130.55, 17.24, 14.70, 12.88, 11.08.19 F NMR (400 MHz, CDCl 3 ): δ = -140.78 (dd, J = 10.09 Hz, J = 21.81 Hz, 2F), -146.73 (dd, J = 32.46 Hz, J = 65.26 Hz, 2F), -151.33 (dd, J = 10.22 Hz, J = 21.81 Hz, 2F), HRMS (ESI): Calc. m/z = 516.1775 for C 23 H 22 BN 5 F 6 Na Found m/z = 516.1764 [M+Na] + triazole-BODIPY (65) azide-BODIPY (25 mg, 51.6 µmol) and [Cutren(CH 2 PhtBu) 3 ](kt) 2(0.58 mg, 0.5 µmol) are dissolved in toluene (0.75 mL) and dodec-1-yne (12 µL, 55.6 µmol) was added in a NMR tube. The solution was bubbled with argon during 20 min, sealed and irradiated at 365 nm with a TLClamp. After 28.5 h the reaction had reached 78 % completion. The volatiles were evaporated and crude product was purified by column chromatography using PTE/DCM (80/20, v/v). The 20 mg (31 µmol, ) of product was obtained as a red crystalline solid.

1 H

 1 NMR (300 MHz, CDCl 3 ): δ = 9.78 (s, 1H), 7.38 (dd, J = 1.73 Hz, J = 5.88 Hz, 1H), 6.88 -6.82 (m, 1H), 6.77 -6.72 (m, 2H), 6.67 -6.58 (m, 2H), 4.33 -4.26 (m, 4H), 4.23 (s, 4H), 4.11 -4.00 (m, 12H), 2.25 (s, 3H), 1.18 -1.12 (m, 12H).Spectra conform with literature BAPTA-BODIPY ester(71) In a two neck round bottom flask under N 2 , BAPTA carboxaldehyde (0.4 g, 0.63 mmol) was dissolved in freshly distilled dichloromethane (80 mL). 2,4-dimethylpyrrole (0.2 mL, 1.9 mmol) and a drop of TFA were added and the mixture was stirred at room temperature overnight. p-Chloranil (0.16 mg, 0.63 mmol) in DCM (20 mL) was added dropwise and the mixture was stirred for 30 min at room temperature. Then

O

  HRMS (FD): Calc. m/z = 736.2727 for C 36 H 39 BF 2 N 4 O 10 Found m/z = 736.2718 [M] +

  Found m/z = 296.0564 [M-H] - 5-(Benzyloxy)-2-(1,3-dioxan-2-yl)-6-nitrobenzofuran (89) 5-(benzyloxy)-6-nitrobenzofuran-2-carbaldehyde (1.4 g, 4.7 mmol), propane diol (5 mL) and p-TsOH monohydrate (0.2 g, 1.05 mmol)

(

  41 mg, 0.29 mmol) was added and the reaction was refluxed for 6 h. After cooling, the reaction was filtered and the filtered product was washed with DCM. The combined organic phases were dried over MgSO 4 , filtered and evaporated. The crude product was purified using PTE/EtAC (70/30, v/v) to yield 120 mg (0.1 mmol, 66 %) of product.

3 ( 3 , 4 -

 334 250 µmol) and a drop of water was added and the mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure and the crude product was dispersed in ethyl acetate and centrifuged. The solvent was pipetted off, the solid was dissolved in water and acidified with 1M HCl. The precipitate was dissolved in an appropriate amount of KOH solution to form the potassium salt and freeze dried to yield the title compound quasi-quantitatively.HRMS (FD):Calc. m/z = 1133.1566 for C 40 H 34 F 17 N 4 O 13 S Found m/z = 1133.1571 [M] + ester (20 mg, 35 µmol) was dissolved in a mixture of THF (1 ml) and MeOH (1 ml), then LiOH.H 2 O (10 mg, 250 µmol) and a drop of water was added and the mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure and the crude product was dispersed in ethyl acetate and centrifuged. The solvent was pipetted off and the solid was dissolved in water and acidified with 1M HCl. The precipitate was dissolved in an appropriate amount of KOH-solution to form the potassium salt and freeze dried to yield the title compound quasi-quantitatively. HRMS (FD): Calc. m/z = 583.1631 for C 22 H 30 N 3 O 14 Na Found m/z = 583.1625 [M-H+Na] + 3,4-dimethoxybenzaldehyde (105) Dimethoxyphenyl)methanol (1 g, 5.95 mmol), potassium iodide (0.25 g, 1.50 mmol), iodine (1.52 g, 5.99 mmol) and K 2 CO 3 (1.24 g, 8.97 mmol)

1 H

 1 NMR (300 MHz, CD 3 Cl): δ = 9.85 (s, 1H), 7.46 (dd, J = 1.87 Hz, J = 8.16 Hz , 1H), 7.42 (d, J = 1.87 Hz, 1H), 6.98 (d, J = 8.16 Hz, 1H), 3.97 (s, 3H), 3.97 (s, 3H). Spectra conform with literature Kamimura, A.; Komatsu, H.; Moriyama, T.; Nozaki, Y. Tetrahedron 2013, 69, 5968.4,5-dimethoxy-2-nitrobenzaldehyde (106)3 3,4-dimethoxybenzaldehyde (2 g, 12 mmol) was added at 0 °C, over a period of 5 min to a cooled solution of HNO 3 (65 % in water). The reaction for 10 min at 0 °C followed by 60 min at room temperature. The reaction mixture was poured into an ice-cooled aqueous solution and filtered. The yellow solid was recrystallised from ethanol to give 2.3 g (90 % yield) of product as a yellow powder. 1 H NMR (200 MHz, CD 3 Cl): δ = 10.45 (s, 1H), 7.61 (d, J = 3.90 Hz, 1H), 7.42 (d, J = 3.93 Hz, 1H), 4.03 (s, 3H), 4.01 (s, 3H). Spectra conform with literature Gavara, L.; Boisse, T.; Henichart, J. P.; Daich, A.; Rigo, B.; Gautret, P. Tetrahedron 2010, 66, 7544.2-(4,5-dimethoxy-2-nitrophenyl)oxirane (107)3

1 H

 1 NMR (300 MHz, CD 3 Cl): δ = 7.73 (s, 1H), 7.04 (s, 1H), 4.53 (dd, J = 2.65 Hz, J = 4.32 Hz, 1H), 3.97 (s, 3H), 3.95 (s, 3H), 3.29 (dd, J = 4.48 Hz, J = 5.49 Hz, 1H), 2.64 (dd, J = 2.63 Hz, J = 5.53 Hz, 1H).

13 C

 13 NMR (75 MHz, CDCl 3 ): δ = 154.36, 148.32, 140.25 129.91, 108.11, 107.91, 56.65, 56.55, 51.32, 50.68. 2-(2-chloroethoxy)ethoxy)-2-(4,5-dimethoxy-2-nitrophenyl)ethanol (108)

1 H

 1 NMR (200 MHz, CD 3 Cl): δ = 7.64 (s, 1H), 7.27 (s, 1H), 5.25 (dd, J = 3.18 Hz, J = 7.31 Hz, 1H), 3.99 (s, 3H), 3.95 (s, 3H), 3.57-3.79 (m, 11H).

13 C

 13 NMR (75 MHz, DMSO): δ = 109.92, 108.11, 79.12, 72.43, 71.46, 71.28, 70.58, 69.15, 66.55, 61.89, 56.68, 56.53, 56.50, 42.86. HRMS (ESI): Calc. m/z = 372.0805 for C 14 H 20 NO 7 ClNa Found m/z = 372.0820 [M+Na] + 1-(1-(2-(2-chloroethoxy)ethoxy)-2-iodoethyl)-4,5-dimethoxy-2-nitrobenzene (109) PPh 3 (2.00 g, 7.62 mmol) was added in one portion to a stirred solution of 1-(1-(2-(2-chloroethoxy)ethoxy)-2-iodoethyl)-4,5-dimethoxy-2nitrobenzene (1.80 g, 5.15 mmol) and imidazole (880 mg, 12.93 mmol) in toluene. I 2 (1.95 g, 7.68 mmol) was added and the reaction mixture was refluxed for 2 h, then the reaction was cooled and diluted with EtAc. The organic phase was washed with saturated Na 2 S 2 O 3 -solution until the iodine color disappeared, followed by 2 washings with brine and water. The organic phase was dried over MgSO 4 , filtered and the solvent evaporated. The crude product was purified by column chromatography PTE/EtAc (60/40, v/v) to obtain 1.1 g (48 % yield) of the product as a yellow oil. 1 H NMR (300 MHz, CD 3 Cl): δ = 7.64 (s, 1H), 7.30 (s, 1H), 5.19 (dd, J = 2.79 Hz, J = 8.07 Hz, 1H), 4.01 (s, 3H), 3.96 (s, 3H), 3.81-3.74 (m, 3H), 3.70-3.56 (m, 6H) 3.33-3.28 (m, 1H).

13 C

 13 ): Calc. m/z = 481.9849 for C 14 H 19 NO 6 ClNa Found m/z = 481.9837 [M+Na] + 2-(2-(2-aminoethoxy)ethoxy)-2-(4,5-dimethoxy-2-nitrophenyl)ethanamine (110) 1-(1-(2-(2-chloroethoxy)ethoxy)-2-iodoethyl)-4,5-dimethoxy-2nitrobenzene (0.85 g, 1.92 mmol) was dissolved in DMF (4 mL) and sodium azide (0.28 g, 4.3 mmol) was added. The reaction mixture was heated to 90 °C and stirred for 5 h. The solvent was evaporated under reduced pressure, the residue dissolved in EtAc and washed with water and brine. The organic phase was dried over MgSO 4 , filtered and the solvent evaporated. To the crude product was added THF (8 ml) PPh 3 (1.05 g, 4.00 mmol) and H 2 O (200 µl). The reaction mixture was stirred at 40 °C over night. The solvent was evaporated and the crude mixture purified by column chromatography using DCM/MeOH/TEA (80/20/1, v/v/v) to yield 0.50 mg (1.52 mmol, 79 %) product as a light yellow liquid. 1 H NMR (300 MHz, CD 3 Cl): δ = 7.63 (s, 1H), 7.24 (s, 1H), 5.08 (dd, J = 2.91 Hz, J = 7.85 Hz, 1H), 3.98 (s, 3H), 3.95 (s, 3H), 3.72-3.51 (m, 4H), 3.49 (t, J = 5.16, 2H), 3.06 (dd, J = 2.93, J = 13.59, 1H), 2.87 (t, J = 5.30, 2H), 2.80 (dd, J = 7.87, J = 13.59, 1H). 13 C NMR (75 MHz, CD 3 Cl): δ = 153.94, 148.10, 140.84, 132.13, 109.39, 108.06, 80.61, 73.47, 70.46, 69.18, 56.59, 56.48, 48.72, 41.81. HRMS (ESI): Calc. m/z = 352.1493 for C 14 H 23 N 3 O 6 Na Found m/z = 352.1479 [M+Na] + DMNPE-4 ester (111) In a round bottom flask, 2-(2-(2-aminoethoxy)ethoxy)-2-(4,5dimethoxy-2-nitrophenyl)ethanamine (0.50 g, 1.52 mmol) and ethyl bromoacetate (1.68 mL, 15.2 mmol) were dissolved in AcCN (15 mL), then Na 2 HPO 4 (0.87 g, 6.12 mmol) and NaI (0.11 g, 0.74 mmol) were added. The reaction mixture was heated to reflux over night. The reaction was cooled to room temperature and the solids filtered off. The solvent was removed under reduced pressure and the crude product chromatography using PET/EtAc (30/70, v/v) to obtain 0.76 g (1.12 mmol, 74 % yield) of the shown compound as a light yellow oil. 1 H NMR (300 MHz, CD 3 Cl): δ = 7.61 (s, 1H), 7.28 (s, 1H), 5.29 (dd, J = 2.67 Hz, J = 7.20 Hz, 1H), 4.22-4.07 (m, 8H), 3.99 (s, 3H), 3.93 (s, 3H), 3.75 (s, 4H), 3.58 (s, 4H) 3.56-3.41 (m, 6H), 3.11 (dd, J = 2.68, J = 14.72, 1H), 3.03 (dd, J = 6.93, J = 14.14, 1H), 2.92 (t, J = 5.77, 2H), 1.30-1.20 (m, 12H) NMR (75 MHz, CD 3 Cl): δ = 171.

4 -

 4 (benzyloxy)-3-methoxybenzaldehyde (113) Vanilin (3.00 g, 19.72 mmol) and benzyl bromide (3.51 ml, 29.55 mmol) were dissolved in acetone (20 mL). K 2 CO 3 (4.08 g, 29.55 mmol) was added and the reaction mixture heated at reflux for 5 h. The solution was cooled to room temperature and filtered. The solid was washed with acetone several times and the combined organic phases were evaporated. The crude product was purified by column chromatography using PTE/DCM (40/60, v/v) to yield 3.8 g (15.7 mmol, 80 % yield).1 H NMR (300 MHz, CD 3 Cl): δ = 9.83 (s, 1H), 7.46-7.32 (m, 7H), 6.99 (d, J = 8.17, 1H), 5.25 (s, 2H), 3.95 (s, 3H).

13 C 4 -

 134 NMR (75 MHz, CD 3 Cl): δ = 190.95, 153.70, 150.19, 136.13, 130.42, 128.81, 128.29, 127.30, 126.63, 112.53, 109.52, 70.97.56.15 HRMS (ESI): Calc. m/z = 243.1019 for C 15 H 15 O 3 Found m/z = 243.1015 [M+H] + 4-(benzyloxy)-5-methoxy-2-nitrobenzaldehyde (114) Benzyloxy-3-methoxybenzaldehyde (3.7 g, 15.3 mmol) was solubilized in acetic acid (6 mL) and cooled in an ice bath. HNO 3 65 % in water (20 mL) was added dropwise over 10 min. After the addition the mixture was stirred at room temperature for 1 h until all starting material was consumed. The reaction mixture was poured into an ice water mixture and the precipitated solid was filtered off and washed with H 2 O. The crude product was purified by column chromatography using gradient elution starting from PTE/DCM (40/60, v/v) to pure DCM yielding 3 g (10.4 mmol, 68% yield) of a yellowish solid. 1 H NMR (300 MHz, CD 3 Cl): δ = 10.43 (s, 1H), 7.66 (s, 1H), 7.49-7.35 (m, 6H), 5.27 (s, 2H), 4.01 (s, 3H).

13 C

 13 NMR (75 MHz, CD 3 Cl): δ = 187.84, 153.86, 151.54, 143.74, 134.99, 129.02, 128.82, 127.70, 125.85, 110.16, 109.04, 71.69, 56.84. HRMS (ESI): Calc. m/z = 288.0866 for C 15 H 14 NO 5 Found m/z = 288.0866 [M+H] +2-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)oxirane (115) A suspension of NaH (275 mg, 11.47 mmol) in DMSO (20 ml) was prepared and stirred under an argon atmosphere for 30 min at 60 °C.The mixture was diluted with THF (20 mL) and cooled to 0 °C. Trimethylsulfonium iodide (1.72 g, 8.43 mmol) in DMSO (5 mL) was added dropwise and subsequently 4-(benzyloxy)-5-methoxy-2-nitrobenzaldehyde (2.20 g, 7.66 mmol) dissolved in THF (5 mL) was added dropwise. After 15 min, another portion of NaH (275 mg, 11.47 mmol) was added. The reaction progression was monitored by TLC PET/EtAc (70/30, v/v). Once the reaction was complete water was added (150 mL) and the mixture was extracted with Et 2 O (3x). The ether phase was washed with brine (2x) and water (2x). The organic phase was dried over MgSO 4 and after filtration, the solution was concentrated to approximately 10 mL and passed through a silica plug eluting with Et 2 O.After evaporation of the solvent in vacuo 1.82 g (6.04 mmol, 79 % yield) of product was obtained.

2-( 2 -

 2 (2-aminoethoxy)ethoxy)-2-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)ethanamine (118) Sodium azide (0.60 g, 9.23 mmol) and 1-(benzyloxy)-4-(1-(2-(2chloroethoxy)ethoxy)-2-iodoethyl)-2-methoxy-5-nitrobenzene(1.30 g, 2.43 mmol) were dissolved in DMF (6 mL) and stirred for 6 h at room temperature so that double substitution could be observed on the TLC PTE/EtAc (80/20, v/v). The solution was cooled, diluted with EtAc and washed with brine (2x) and water (1x). The organic phase was dried over MgSO 4 , filtered and the volatiles were evaporated. To the crude product was solubilized in THF (10 ml) and H 2 O (200 µL), subsequently PPh 3 (1.32 g, 5.03 mmol) was added and the reaction mixture was stirred at room temperature overnight. The solvent was evaporated and the product was purified by column chromatography using DCM/MeOH/TEA (90/10/1, v/v/v) to yield 0.82 g (2.02 mmol, 83 % yield) of product as a yellowish oil.

1 H

 1 NMR (300 MHz, CDCl 3 ): δ = 7.64 (s, 1H), 7.29 (s, 1H), 5.22 (dd, J = 2.28, J = 7.48, 1H), 4.21-4.08 (m, 8H), 4.02 (s, 3H), 3.74 (s, 4H), 3.58 (s, 4H), 3.57-3.39 (m, 6H), 3.11 (dd, J = 2.27, J = 14.61, 1H), 3.04-2.90 (m, 3H), 1.30-1.21 (m, 12H).

13 C

 13 NMR (75 MHz, CDCl 3 ): δ = 172.04, 171.47, 144.83, 141.20, 111.37, 109.59, 70.41, 68.87, 61.40, 60.59, 60.51, 56.78, 55.88, 53.72, 21.14, 14.36, 14.32. HRMS (ESI): Calc. m/z = 682.2786 for C 36 H 51 N 3 O 14 Na Found m/z = 682.2793 [M+Na] + C 8 F 17 -DMNPE-4 ester (121)In a round bottom flask HO-DMNPE-4-ester (100 mg, 0.15 mmol) and heptadecafluoro-1-dodecanol (100 mg, 0.18 mmol) were dissolved in AcCN (3 mL). Cs 2 CO 3 (98 mg, 0.30 mmol) was added and the reaction mixture was stirred at room temperature overnight. The solution was filtered and the solvent was evaporated under reduced pressure. The crude product was purified by column chromatography using PTE/EtAc (70/30, v/v) which yielded 100 mg (0.09 mmol, 58 %) of product as a yellow oil.

  (300 MHz, CDCl 3 ): δ = 7.59 (s, 1H), 7.27 (s, 1H), 5.29 (dd, J = 2.73, J = 7.08, 1H), 4.20-4.06 (m, 10H), 3.96 (s, 3H), 3.75 (s, 3H), 3.58 (s, 4H), 3.56-3.38 (m, 6H), 3.10 (dd, J = 2.60, J = 14.69, 1H), 3.04-2.89 (m, 3H), 2.35-2.08 (m, 2H), 2.04-1.75 (m, 4H), 1.30-1.20 (m, 12H).

13

 13

  .

19 F

 19 NMR (400 MHz, CDCl 3 ): δ = -81.72 (t, J = 9.98 Hz, 3F), -115.35-115.52 (m, 2F), -122.65 (s, br, 2F), -122.87 (s, br, 4F), -123.68 (s, br, 2F), 124.46 (s, br, 2F), 127.04 (s, br, 2F). HRMS (ESI): Calc. m/z = 1133.3136 for C 41 H 52 F 17 N 3 O 14 Found m/z = 1133.3178 [M+H] +C 8 F 17 -DMNPE-4 (122) C 8 F 17 -DMNPE-4 ester (20 mg, 18 µmol) was dissolved in a mixture of THF (1 mL) and MeOH (1 mL), then LiOH.H 2 O (10 mg, 250 µmol) and a drop of water was added and the mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure and the crude product was dispersed in ethyl acetate and centrifuged. The solvent was pipetted off, the solid dissolved in water and acidified with 1M HCl. The precipitate was dissolved in an appropiate amount of KOH solution to form the potassium salt and freezedried to yield the title compound quasi-quantitatively. MS (MALDI): Calc. m/z = 1173.0 for C 31 H 32 F 17 N 3 O 14 K Found m/z = 1173.6 [M] + (Potassium salt) µL, 0.36 mmol) were dissolved in AcCN (5 ml) and Cs 2 CO 3 (195 mg, 0.6 mmol) was added. The reaction mixture was refluxed for 1 h, cooled to room temperature and filtered. The solvent was evaporated under reduced pressure and the crude product was purified by column chromatography using PTE/EtAc (80/20, v/v) to yield 180 mg (0.21 mmol, 70 %) of product as a light yellow oil.

C 14 H

 14 29 -DMNPE-4 (124) C 14 H 29 -DMNPE-4-ester (15 mg, 18 µmol) was dissolved in a mixture of THF (1 mL) and MeOH (1 mL), then LiOH.H 2 O (10 mg, 250 µmol) and a drop of water was added and the mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure and the crude product was dispersed in ethyl acetate and centrifuged. The solvent was pipetted off, the solid was dissolved in water and acidified with 1M HCl. The precipitate was dissolved in an appropiate amount of KOH solution to form the potassium salt and freezedried to yield the title compound quasi-quantitatively.

  /z = 782.3478 for C 35 H 57 N 3 O 14 K Found m/z = 782.34832 [M+K] + Heptadecafluoro-3-iodododecan-1-ol (135) 2

19 F

 19 NMR (400 MHz, CDCl 3 ): δ = -159.04 --159.12 (m, 2F), -152.40 (t, J = 21.73 Hz, 1F), -146.61 (q, J = 30.72 Hz, 2F), -138.40 --138.69 (m, 2F). HRMS (ESI): Calc. m/z = 493.1667 for C 23 H 22 BN 2 F 7 Na Found m/z = 493.1656 [M+Na] + C 8 F 17 -BODIPY (149)F5-BODIPY (80 mg, 0.17 mmol) and heptadecafluorodecane-1-thiol (97 mg, 0.20 mmol) were dissolved in AcCN (5 mL) and TEA (86 µL, 0.6 mmol) was added. The reaction mixture was stirred at room temperature for 2 h. The volatiles were evaporated under reduced pressure and the crude product was purified by column chromatography using PTE/DCM (80/20, v/v) to yield 100 mg (0.11 mmol, 63 %) of product as a red powder.

13 CBODred

 13 (300 MHz, CDCl 3 ): δ = 3.22-3.14 (m, 2H), 2.50 (s, 6H), 2.40-2.23 (m, 6H), 1.45 (s, 6H), 0.97 (t, J = 7.58 Hz, 6H). NMR (75 MHz, CDCl 3 ): δ = 156.24, 136.60, 134.05, 130.19, 17.23, 14.63, 12.91, 10.78.19 F NMR (400 MHz, CDCl 3 ): δ = -81.68 (t, J = 9.91 Hz, 3F), -114.88 --115.04 (m, 2F), -122.60 (s, 2F), -122.83 (s, 4F), -123.64 (s, 2F), -124.37(s, 2F), -127.04 (2F), -132.07 (dd, J = 12.44 Hz, J = 24.40 Hz, 2F), -139.57 (dd, J = 12.47 Hz, J = 24.35 Hz, 2F), -146.73 (q, J = 32.47 Hz, 2F). HRMS (ESI): Calc. m/z = 929.1623 for C 33 H 26 B N 2 F 23 S Found m/z = 929.1579 [M+H] + mg, 0.048 mmol) were dissolved in toluene (2 mL) and piperidine (6.3 µL, 0.064 mmol) was added.

  ): Calc. m/z = 1108.2282 for C 43 H 36 B F 23 N 2 O 3 S Found m/z = 1107.22087 [M-H] + C 8 F 17 -fluorescein (152) Fluorescein (130 mg, 0.39 mmol) and C 8 F 17 -butyl bromide (242 mg, 0.45 mmol) were dissolved in DMF (2 mL). The reaction mixture was stirred overnight at 60 °C. The solvent was evaporated and the crude product was purified by column chromatography starting with pure DCM to DCM/MeOH (90/10, v/v) to yield 40 mg (0.05 mmol, 12 %) of product. 1 H NMR (400 MHz, CD 3 OD): δ = 8.29 (dd, J = 1.41 Hz, J = 7.78 Hz, 1H), 7.86 -7.76 (m, 2H), 7.43 (dd, J = 1.31 Hz, J = 7.53 Hz, 1H), 7.01 (d, J = 9.20 Hz, 2H), 6.74 (d, J = 2.06 Hz, 2H), 6.69 (dd, J = 2.16 Hz, J = 9.22 Hz, 2H), 4.01, (t, J = 5.84 Hz, 2H), 2.09 -1.96 (m, 2H), 1.47 -1.29 (m, 5H).

13 C

 13 NMR (75 MHz, CD 3 OD): δ = 166.86, 159.03, 156.57, 135.19, 134.02, 132.23, 131.94, 131.74, 131.24, 116.38, 104.33, 65.92, 31.12, 30.90, 28.80, 18.00. 19 F NMR (400 MHz, CD 3 OD): δ = -83.32 (t, J = 10.15 Hz, 3F), -116.27 --116.44 (m, 2F), .123.61 (s, 2F), -123.84 (s, 4F), -124.68 (s, 2F), -125.46 (s, 2F), -128.22 (s, 2F). HRMS (FD): Calc. m/z = 806.0992 for C 32 H 19 F 17 O 5 Found m/z = 806.0961 [M] + 2,4-Dihydroxy-3,5-difluoro benzaldehyde (154) 7

  (300 MHz, CDCl 3 ): δ = 9.63 (d, J = 1.93 Hz, 1H), 7.03 (dd, J = 2.06 Hz, J = 9.76 Hz, 1H). Spectra conform with literature Gee, Kyle R.; Haugland, Richard P.; Sun, Wei Chuan. UK Pat. Appl. (1998), GB 2319250 A 19980520

1 H 13 C

 113 NMR (400 MHz, DMSO-d 6 ): δ = 8.50 (s, br, 1H), 8.20 (s, 1H), 7.18 (d, J = 11.49 Hz, 1H). NMR (75 MHz, CDCl 3 ): δ = 171.76, 164.13, 149.25, 142.70, 142.59, 108.79, 108.62, 99.81, 99.73, 28.73. 19 F NMR (400 MHz, CDCl 3 ): δ = -74.51 (s, br, 1F), -136.73 (s, br, 1F) HRMS (FD): Calc. m/z = 242.0020 for C 10 H 4 F 2 O 5 Found m/z = 242.0027 [M] + Pacific-C 8 F 17 (156) Pacific blue (20 mg, 0.076 mmol) and HBTU (86 mg, 0.228 mmol)were dissolved in THF (2 mL) and diisopropylethylamine (100 µL) was added. The reaction was stirred for 5 h at room temperature and subsequently water and DCM were added. The aqueous phase was extracted with DCM (3x) and the organic phase washed with brine (2x), HCl (1 N, 2x) and water (2x). The organic phase was dried over MgSO 4 , filtered and evaporated. The crude product was purified by column chromatography using DCM/MeOH (90/10, v/v) to yield 17 mg (0.025 mmol, 33 %) of the product as a yellowish powder.

99 F 17 C 8

 99178 photocontrolés dans compartiments confiné Cette thèse se focalise sur la synthèse et l'étude de nouvelles molécules photoactives et leurs applications en tant que marqueurs, senseurs moléculaires et récepteurs d'ions photomodulables en milieux aqueux et organisés. Les fluorophores développés sont principalement des dérivés du bore-dipyrométhene (BODIPY), comportant des groupements réactifs (azoture, perfluorophényle), des chaines hydrophobes, ou sont intégrés à un récepteur de calcium biocompatible. Le développement d'architectures auto-assemblées multicompartimentées de type vésicules dans des polymersomes géant y est décrit. Ces architectures ont été utilisées pour la génération de lumière blanche dans un micro-domaine, et constitue un modèle pour l'étude de transfert d'ions calcium entre vésicules localisées dans des polymersomes individuels. Ce transfert entre nano-objets confinés à l'intérieur d'un polymersome géant représente un système prototype de communication cellulaire artificiel rudimentaire.

  application as labeling agents, fluorescent molecular Ca 2+ -sensors and photolabile Ca 2+decaging agents in aqueous media and organized supramolecular assemblies. The designed fluorophores are based on boron-dipyrromethene (BODIPY) bearing hydrophobic chains or a reactive group like an azide or a perfluorophenyl moiety. Biocompatible calcium receptors have been prepared harnessing the fluorescence properties of BODIPY, naphthalimide and furan fluorophores. The development of self-assembled multicompartmentalized architectures, namely fluorocarbon vesicles in giant polymersomes is reported and the system has been used to create white light emission in confined microdomains. The Ca 2+ -based ion transfer ion the confined polymer compartments between individual fluorinated vesicles has been studied. The ion transfer in between vesicles in polymer microcompartments has been established as an artificial prototype system for cellular communication. Keywords: Supramolecular chemistry, self-assembly, photochemistry, multicompartmentalization, liposome, polymersome, ion receptor, fluorescence, ion decaging.

  Ions in biology ............................................................................................................ 1.2 Goal of the project ...................................................................................................... 1.3 Supramolecular chemistry ..........................................................................................

	1.1

  1

Table 01 :

 01 Comparison of ion concentration inside and outside of mammalian cells.2

	Cation	Intracellular	Extracellular
		concentration (mM)	concentration (mM)
	Na +	5 -15	145
	K +	140	5
	Mg 2+	0.5	1 -2
	Ca 2+	10 -4	1 -2
	H +	7 x 10 -5 (10 -7.2 M or pH 7.2)	4 x 10 -5 (10 -7.4 M or pH 7.4)
	Anion
	Cl -	5 -15	110

Table 02 :

 02 Strength of molecular interactions.17

	Interaction	strength
	Covalent bond	145-1070 kJ/mol
	Ion-ion	100-350 kJ/mol
	Ion-dipole	50-200 kJ/mol
	Dipole-dipole	5-50 kJ/mol
	Hydrogen bond	4-60 kJ/mol
	Ion-π	38-159 kJ/mol
	π-π	0-50 kJ/mol
	Hydrophobic	< 8 kJ/mol

Table 04 :

 04 Absorption and emission maxima of BAPTA PCT sensors in presence and absence of Ca 2+ .

		Abs. max (nm)	Abs. max (nm)	Em. max (nm)	Em. max (nm)
		(-Ca 2+ )	(+Ca 2+ )	(-Ca 2+ )	(+Ca 2+ )
	Indo-1 (34)	349	331	485	410
	Fura-2 (35)	362	335	512	505
	Still-1 (36)	362	329	537	529

Table 04 :

 04 Spectral properties of 59 -62 in air equilibrated THF

Molecule λ max (nm) ε (M -1 cm -1 ) λ em (nm) Ф f ז calc (ns) ז (ns)

	the wavenumber (ν -3 ) across the fluorescence spectrum, ε is the molar absorptivity and the ) across the fluorescence spectrum, ε is the molar absorptivity and the ) across the fluorescence spectrum, ε is the molar absorptivity and the
	natural lifetime (τ rad ). 27
							equation 07
	Substituting the reciprocal of the natural lifetime (k Substituting the reciprocal of the natural lifetime (k r ) into equation 08 gives estimated into equation 08 gives estimated
	fluorescence lifetimes on the nanosecond scale which are in good agreement with the fluorescence lifetimes on the nanosecond scale which are in good agreement with the fluorescence lifetimes on the nanosecond scale which are in good agreement with the
	measured values (table 04).
	59	516	78600	527	0.98	5.8	6.1 equation 08
	60 Figure 40: Measured transient absorption spectrum (TRABS, black) in THF. Steady 516 77000 527 0.90 5.4 Measured transient absorption spectrum (TRABS, black) in THF. Steady	5.9
	61 This suggests that the excited parent and derivativ 514 78700 525 This suggests that the excited parent and derivatives are all structurally reminiscent to that of 0.93 6.1 5.7 es are all structurally reminiscent to that of absorption (red) and steady state fluorescence (green). The excited state spectrum of absorption (red) and steady state fluorescence (green). The excited state spectrum of 59
	62 the respective ground-state species and that solvent interactions are unchanging. 515 77200 523 0.96 6.0 state species and that solvent interactions are unchanging. state species and that solvent interactions are unchanging. subtracting absorption, fluorescence/stim subtracting absorption, fluorescence/stimulated emission contributions from TRABS (λ exc	5.7
	Fluorescence quantum yields and lifetimes have been measured in a mixture of H (75/25, v/v) to show the feasibility Fluorescence quantum yields and lifetimes have been measured in a mixture of H (75/25, v/v) to show the feasibility of labeling applications and fluorescence measurements in A similar excited state absorption has been obtained for a multicomponent BODIPY A similar excited state absorption has been obtained for a multicomponent BODIPY Fluorescence quantum yields and lifetimes have been measured in a mixture of H 2 O and DMF of labeling applications and fluorescence measurements in containing molecule 29 . The excited state spectrum, which is close to the ground state . The excited state spectrum, which is close to the ground state
	aqueous media. DMF needed to be added to increase the solubility of the test compounds. aqueous media. DMF needed to be added to increase the solubility of the test compounds. aqueous media. DMF needed to be added to increase the solubility of the test compounds. absorption spectrum leads to t absorption spectrum leads to the conclusion that no significant population of states other than
	Similarly high quantum yields have been measured for BODIPY Similarly high quantum yields have been measured for BODIPY 59 and 60 60, which have been the emissive S 1 state occur.
	determined to be 0.91 with a lifetime of 6.6 ith a lifetime of 6.6 ns and 0.93 with a lifetime of 6.6 ns and 0.93 with a lifetime of 6.6 ns, respectively.
	Meanwhile, the amine derivative ( Meanwhile, the amine derivative (61) exhibits a drastically reduced quantum yield of 1 % ) exhibits a drastically reduced quantum yield of 1 % The quantum yield of photodegradation had been determined taking in account the reaction The quantum yield of photodegradation had been determined taking in account the reaction
	presumably due to hydrogen bonding enhanced vibrational quenching. This principle is presumably due to hydrogen bonding enhanced vibrational quenching. This principle is presumably due to hydrogen bonding enhanced vibrational quenching. This principle is scheme presented in figure 41. It was assumed that all excited molecule scheme presented in figure 41. It was assumed that all excited molecules relax to the ground
	exploited in BODIPY-based pH sensors carrying amines, for example based pH sensors carrying amines, for example. 28 Picosecond transient Picosecond transient state, as supported by the transient absorption measurements shown earlier, except a small state, as supported by the transient absorption measurements shown earlier, except a small
	absorption spectroscopy has been employed to follow the evolution of the excited state of absorption spectroscopy has been employed to follow the evolution of the excited state of absorption spectroscopy has been employed to follow the evolution of the excited state of population which is devoted to the quantum yield of photo degradation (Q population which is devoted to the quantum yield of photo degradation (Q Photoproduct
	parent F 5 -BODIPY (59) to elucidate the presence of any dark ) to elucidate the presence of any dark-state, which could not state, which could not be
	detected by classical fluorescence spectroscopy. Population of such a state would present an detected by classical fluorescence spectroscopy. Population of such a state would present an detected by classical fluorescence spectroscopy. Population of such a state would present an
	unwanted alternative to emissive deexcitation and could promote degradation of the molecule. unwanted alternative to emissive deexcitation and could promote degradation of the molecule. unwanted alternative to emissive deexcitation and could promote degradation of the molecule.
	The transient absorption spectrum of the excited state are plotted The transient absorption spectrum of the excited state are plotted in figure 40, along with in figure 40, along with
	fluorescence and absorption spectra for comparison. The transient absorption spectrum fluorescence and absorption spectra for comparison. The transient absorption spectrum fluorescence and absorption spectra for comparison. The transient absorption spectrum Figure 41:
	(TRABS, black) resembles the fluorescence emission spectra (FLUO, green) with only minor (TRABS, black) resembles the fluorescence emission spectra (FLUO, green) with only minor (TRABS, black) resembles the fluorescence emission spectra (FLUO, green) with only minor
	intensity differences. Two positive transient absorption intensity differences. Two positive transient absorption bands have been observed at 350 bands have been observed at 350 nm As stated by the Stark-Einstein law, each photon absorbed by a chemical system, only one
	and 420 nm while a larger negative band appears at 525 nm while a larger negative band appears at 525 nm. By subtracting the fluorescence nm. By subtracting the fluorescence molecule is activated for a following reaction. According to this law of photo equivalence
	emission spectra (green) and the absorption spectrum (ABS, red) from TRABS, the excited emission spectra (green) and the absorption spectrum (ABS, red) from TRABS, the excited emission spectra (green) and the absorption spectrum (ABS, red) from TRABS, the excited equation 09 can be formulated and shows that the number of molecules excited (N
	state spectrum shown in blue is obtained. state spectrum shown in blue is obtained. the number of photons absorbed (K
				52 53

  3.1.4 Spectral characterization of BAPTA-BODIPY (72 and 76) and their corresponding esters (71 and 75) The ester-protected BAPTA-BODIPY molecules (71 and 75) were studied in air equilibrated THF by steady-state electronic absorption and emission spectroscopy. The spectral data obtained are summarised in table 05.

Table 05 :

 05 Summary of the spectral characterization of BAPTA-BODIPYs and esters.

		ʎ max	ɛ (M -1 cm -1 )	ʎ em	ɸ em
	71	501	79500	514	0.01
	75	501	82800	512	0.04
	72	498		508	0.0003
	76	497		507	0.004

Table 06 :

 06 Summary of the absorption, emission and complexation characteristics of BAPTA-BODIPYs.

		λ max	λ em	ɸ em	pK d	F max /F min
	72	498	508	0.0003
	76	497	507	0.004
	72+Ca 2+	498	508	0.04	6.3	122
	76+Ca 2+	497	507	0.09	6.8	23
	The calcium dissociation constants (K

d ) were established to be 0.53 µM for BAPTA-BODIPY

(72)

and 0.16 µM for BAPTA-PH-BODIPY

(76)

, showing a similar trend to spectrophotmetrically-determined values. However, it is noteworthy that even though the binding constants for fluorionophore 76 are in good agreement with each other there is a bigger difference in the values obtained for Ca 2+ -sensor 72 and corresponds more to the value of an unsubstituted BAPTA. The difference in the determined K d values from fluorescence and UV-Vis measurements for BAPTA-BODIPY (

72

) is most likely based on the a polar excited state of the molecule which is supported by the appearance of an charge transfer band in the emission spectra seen for the ester (71) in THF. The excited state has a lowered electron density on the aromatic ring which leads to a lower binding affinity.

  in presence of a benzyl protecting group has been shown by Grynkiewicz et. al.1 amongst others. Therefore a platinum catalyst on charcoal was employed under hydrogen atmosphere to yield the selectively reduced diamine intermediate(92) with a reaction yield of 73 %. Several different methods have been employed to alkylate the diamine in order to introduce the ester protected carboxylic acid functions. The diamine intermediate proved to be unstable under reaction conditions involving inorganic salts like K 2 CO 3 and Na 2 HPO 4 .

	deprotection of the benzyl group, it was reacted with 1,3-propanediol to obtain a six-ring
	acetal which is easier to hydrolyse than a 5-membered acetal. 88 was reacted with an excess
	of the diol in the presence of p-toluenesulfonic acid to obtain the benzofuran acetal (89) in
	almost quantitative yield (95 %).

2-methoxy-5-hydroxy benzaldehyde

(83)

was reacted with ethyl bromoacetate under basic conditions. Sufficiently long reaction times allowed the two step reaction, first nucleophilic addition and second cyclisation to yield ethyl 5-methoxybenzofuran-2-carboxylate (84) in 45 % yield.

28

Deprotection of the methoxy group to yield the free alcohol and subsequent reduction of the ester group were achieved in one pot without intermediate purification of the compound to give 2-(hydroxymethyl)benzofuran-5-ol (85) in 48 % over two steps. In the next step the benzyl protecting group was introduced due to a better orthogonality since it could be removed under mild conditions, more precisely catalytic hydrogenation later on in the synthesis. To that end, 85 was reacted with benzylbromide under basic conditions to give the desired protected furan (86) in 67 % yield. Introduction of the nitro group in ortho-position of the protected phenol was achieved by adding HNO 3 (65 %) to a cooled solution of 86 in DCM. Other approaches like classical HNO 3 / acetic acid gave predominantly the substitution in the 3-position of the benzofuran, whereas substitution in the 6-position dominated at low temperatures yielding the desired (5-(benzyloxy)-6-nitrobenzofuran-2-yl)methanol (87) in 63 % yield. Pyridinium chlorochromate was used to oxidize the free benzylic alcohol to an aldehyde following a procedure published by Dauben et. al.

29

. The corresponding aldehyde

(88)

was obtained in 66 % yield. In order to protect the aldehyde from the following catalytic groups

Table 07 :

 07 Summary of the spectral characterization of amphiphilic fluorescent ion switches.

	Sample	ɛ (M -1 cm -1 )	λ abs	λ em	ɸ fluorescence	F Ca /F
	79	17300	436	550	0.006
	79+Ca 2+		436	575	0.026	4.3
	82	56800	529	542	0.001
	82+Ca 2+		529	542	0.004	4
	97	30700	456	672	0.006
	97+Ca 2+		413	672	0.004	0.6

All three amphiphilic fluorescent Ca 2+ -sensors posses interesting properties and could be appropriate sensors for the envisaged ion transfer studies in vesicles. However, before studying the ion transfer in nanodomains, photoactive Ca 2+ -decaging agents need to be chosen for the ion transfer in solution and later on in nanodomains.

3.3 Water soluble and amphiphilic nitrobenzyl-based Ca 2+ -decaging agents

In the general introduction ion binding motives and triggered ion movement / release has been discussed. In this section specific Ca 2+ -decaging agents are introduced and the synthesis and photochemistry of selected Ca 2+ -decaging molecules, appropriate for the following ion transfer experiments are discussed. Many different photolabile Ca 2+ -decaging agents are now known, showing different decaging mechanisms and bearing different photoactive groups. A selection of photoactive molecules described in the literature is given in table 08 and compared to their non-photocleavable molecular counterpart. The nitrobenzyl chromophore is a widely used photo-decaging group and is used, for example, to study ATP-dependent processes in biological systems.

Table 08 :

 08 Properties of photolabile Ca 2+ -cages and Ca 2+ -chelators.

		K d (Ca 2+ )	K d (prod.)	Affinity	K d (Mg)	Quantum	ɛ (cm -1 M -
		(µM)	(µM)	change	(µM)	yield (ɸ)	1 )
	EDTA	0.032			5
	EGTA	0.150			12 000
	BAPTA	0.110			17 000
	DM-nitrophen	0.005	3 x 10 3	600 000	2.5	0.18	4 300
	NP-EGTA	0.080	1 x 10 3	12 500	9 000	0.2-0.23	975
	DMNPE-4	0.048	2 x 10 3	40 000	10 000	0.09	5 120
	nitr-5	0.145	6.3	54	8 500	0.012-0.035	5 500
	azid-1	0.230	0.12 x 10 3	520	8 000	1.0	33 000

  2+ -decaging agents DMNPE-4 (99) and amphiphilic DMNPE-4 (122 and 124) 3.3.1.1 Synthesis of DMNPE-4(99)

Table 09 :

 09 Spectral properties of DMNPE-4 (99) and C 14 H 29 -DMNPE-4 (122).

		Abs. max	ɛ (cm -1 M -1 )	ɸ photocleavage	K d (µM)
	99	345	5110	0.09	0.057
	124	346	5150	0.1	0.032

  Fluorescence emission spectra of a solution containing 0.62 mM of C 14 H 29 -DMNPE-4 (124) and 5.2 µM of BAPTA-BODIPY(72) in the presence of varying amounts of Ca 2+ in pseudo intracellular buffer (λ exc = 476 nm) (left). Scatchard plot of the Ca 2+ -titration (right).The K d value obtained from the linear fitting of the Scatchard plot gave a dissocation constant of K d = 0.032 µM which is slightly lower, but in good agreement with, the K d value of the parent molecule. Since the binding affinity for DMNPE-4 (99) and C 14 H 29 -DMNPE-4 (124)

				0.00 mM Ca 2+
				0.06 mM Ca 2+		16000
				0.12 mM Ca 2+		14000
				0.18 mM Ca 2+
	Fluorescence (A.U.)			0.72 mM Ca 2+ 0.66 mM Ca 2+ 0.24 mM Ca 2+ 0.30 mM Ca 2+ 0.36 mM Ca 2+ 0.42 mM Ca 2+ 0.48 mM Ca 2+ 0.54 mM Ca 0.60 mM Ca 2+ 2+	[Ca 2+ ] bound /[Ca 2+ ] free	2000 12000 4000 6000 8000 10000	R 2 = 0.996 Kd = 0.057 µM
							0
							-2000
	500	550	600	650	700		0.0001	0.0002	0.0003	0.0004	0.0005	0.0006	0.0007
			Wavelength (nm)						[Ca	2+ ] bound
	Figure 69:

Table 10 :

 10 Overview of formulation C -D

		Gemini conc.	Ca 2+ -sensor	Ca 2+ -sensor	Average size
					conc.			DLS
	Formulation C		1.5 mM		12.5 µM		BANIF (79)	138 nm
	Formulation D		1.5 mM		10.1 µM		C 8 F 17 -BAPTA-	134 nm
							BODIPY (82)
	Formulation E		1.5 mM		9.9 µM		C 8 F 17 -Fura red	132 nm
							(97)
	pseudo-intracellular	buffer	containing	either	10 mM	HEEDTA	(N-(2-
	hydroxyethyl)ethylenediamine-N,N',N'-triacetic acid) or 10 mM CaHEEDTA. HEEDTA was
	chosen for the determination of the Ca 2+ -dissociation constants due to its lower binding
	affinity towards Ca 2+ (5.55 at pH 7.24) 37 compared to EGTA and therefore a wider range of
	Ca 2+ concentrations. The fluorescence titration at different concentration of Ca 2+ for
	formulation C is shown in figure 81. The emission maximum shifts from 523 nm to 530 nm
	with increasing amount of Ca 2+ which is somewhat smaller than the shift measured for the
	molecule in solution (550	575 nm)

Formulations C -E were prepared via the thin film hydration method previously described. A mixture of amphiphilic Ca 2+ sensor (79, 82 or 97) and fluorinated gemini (138) in a chloroform / methanol mixture was slowly evaporated and the thin film rehydrated with

Table 11 :

 11 binding affinities and quantum yields of formulation C -D and 79, 82, 97 in aqueous solution.

		Form. C	BANIF (79)	Form. D	C 8 F 17 -BAPTA-	Form E	C 8 F 17 -furared
					BODIPY (82)		(97)
	K d	3.0	0.117	77.6	0.724	9.1	0.076
	ɸ em (+Ca 2+ )	0.28	0.026	0.43	0.004	0.05	0.004
	ɸ em (-Ca 2+ )	0.17	0.006	0.26	0.001	0.08	0.006

Table 12 :

 12 Coordinates of common white light illuminants 3 .

	Illuminant	X	Y	description
	D50	0.34567	0.35850	Horizon Light
	D55	0.33242	0.34743	Mid-morning / Mid-afternoon Daylight
	D65	0.31271	0.32902	Noon Daylight, Television
	D75	0.29902	0.31485	North sky Daylight

Table 13 :

 13 Concentration and size of fluorinated R G B vesicles

	Sample	Concentration of	Concentration of	Mean diameter
		fluorophore	amphiphile
	Formulation R1	3.2 µM	1.5 mM	452 nm
	Formulation G1	20.3 µM	1.5 mM	520 nm
	Formulation B1	4.2 µM	1.5 mM	524 nm

Table 14 :

 14 Spectral characterization of fluorophores in solution and in vesicle membranes. While the fluorescence quantum yield of pacific-C 8 F 17 (156) seems to be unaffected by the incorporation into the vesicle membrane, C 8 F 17 -fluorescein (152) and BODred (150) show a difference. Upon excitation at 375 nm the quantum yield decreases 16 %. Considering that the vesicles themselves absorb a part of the light at 375 nm, that can explain the lower quantum yield. Upon excitation at 458 nm this effect should be negligible and in fact the quantum yield

	Sample	ε	Abs.	Em. max.	Ф	Ф	Ф
		(l mol -1 )	max.		375 nm	458 nm	561 nm
	(1)	27940	412	455	0.97	-
	Form B		408	449	0.96
	(2)	51507	504	526	0.68	0.61
	Form G		509	531	0.52	0.69
	(3)	91228	625	657	0.63	0.54	0.68
	Form R		619	651	0.26	0.22	0.32

Table 15 :

 15 Fluorescence lifetimes (λ exc = 371 nm) of fluorophores 150, 152 and 156 in MeOH (150 in THF) and The longer lifetime component in the membrane of the vesicles displays only little change. The fluorescence lifetime of pacific-C 8 F 17 (156) increases from 3.54 ns to 3.81 ns once incorporated in the membrane with a shorter lifetime component of 2.23 participation indicating that most of the fluorophore is located in the core region. C

	form. R1, G1, B1 and W in Buffer (30 mM MOPS, 100 mM KCl, pH 7.2, 130 mg/ml sucrose)
		(156)	Form. B	(152)	Form. G	(150)	Form. R	Form. W	Form. W	Form. W
		(ns)	(ns)	(ns)	(ns)	(ns)	(ns)	(450 nm)	(540 nm)	(651 nm)
								(ns)	(ns)	(ns)
	ז 1	3.54	3.81	4.88	4.12	5.28	4.38	3.80	4.13	4.34
			(86%)		(80%)		(63%)	(85%)	(67%)	(58%)
	ז 2	-	2.23	-	2.25	-	2.43	2.13	2.53	2.55
			(14%)		(20%)		(36%)	(15%)	(33%)	(42%)

  NMR (75 MHz, CDCl 3 ): δ = 193.54, 157.33, 143.12, 122.17, 78.49, 78.16, 77.83, 44.96, 32.54, 19.72, 14.11, 13.32, 13.03.

	1 H NMR (300 MHz, CDCl 3 ): δ = 6.20 (s, 2H), 4.95 (s, br, 1H), 3.43-3.51 (m, 2H), 2.53 (s,
	6H), 1.73 (s, 6H), 1.58-1.68 (m, 2H), 1.36-1.49 (m, 2H), 0.97 (t, J = 7.3, 3H).

13

C

  H NMR (400 MHz, CDCl 3 ): δ = 7.70 (s, 1H), 2.86 (t, J = 7.71 Hz, 2H), 2.56 (s, 6H), 2.35 (q, was diluted with toluene and washed with water. The organic phase was dried over MgSO 4 , filtered and evaporated to dryness. The crude product was recrystallized from hot EtOH to yield 18.1 g (30.1 mmol, 35 %) of product.

	NO 2	NO 2	5-methyl-2-nitrophenol (30 g, 196 mmol) was solubilized in hot MeOH
	O	O			and a mixture of KOH (11 g, 196 mmol) in MeOH was added to form a
					red precipitate. The solvent was evaporated and the residue was
	J = 7.56 Hz, 4H), 1.81 -1.74 (m, 2H), 1.58 (s, 6H), 1.46 -1.25 (m, 16H), 1.03 (t, J = 7.56 dissolved in DMF (90 mL). 1-(2-Bromoethoxy)-2-nitrobenzene (45 g, 196 mmol) was added 1 H NMR (300 MHz, CDCl 3 ): δ = 6.88 -6.67 (m, 7H), 4.27 (s, 4H), 4.13 (m, 8H), 4.04 (m,
	Hz, 6H), 0.88 (t, J = 6.81 Hz, 3H). and the mixture was stirred at 120°C for 2 h. After cooling to room temperature, the reaction 8H), 2.26 (s, 3H), 1.49 (m, 12H).
	19 F NMR (400 MHz, CDCl 3 ): δ = -183.52 --183.62 (m, 2F), -145.90 --145.98 (m, 2F), -mixture was diluted with water (800 mL) and filtered. The precipitate was dried and Spectra conform with literature
	146.56 --146.82 (m, 2F). recrystallized from hot EtOH containing 5 % water to yield 41.8 g (131.5 mmol, 67 %) of the
	13 C NMR (150 MHz, CDCl 3 ): δ = 156.61, 149.20, 145.36 -145.28 (m), 143.70 -143.62 (m), product as a white powder.
	142.67 (d, J = 14.31 Hz), 140.94 (d, J = 12.76 Hz), 136.59, 134.26, 130.13, 123.02, 120.95, BAPTA-aldehyde (70) 5
	118.37 -118.20 (m), 116.46 (t, J = 19.29 Hz), 32.05, 29.72 (d, J = 7.59 Hz), 29.49 (d, J = 1 H NMR (300 MHz, CDCl 3 ): δ = 7.79 -6.85 (m, 7H), 4.53 (s, 4H), 2.44 (s, 3H)
	4.21 Hz), 29.35 (d, J = 8.73 Hz), 25.84, 22.84, 17.27, 14.70, 14.27, 12.95, 11.23. Spectra conform with literature
	HRMS (FD):				Calc. m/z = 658.3602 for C 35 H 44 BN 5 F 6
						Found m/z = 658.3630 [M] +
	2-(2-(2-aminophenoxy)ethoxy)-4-methylaniline (68) 5
	NH 2	NH 2	4-methyl-1-nitro-2-(2-(2-nitrophenoxy)ethoxy)-benzene
	1-(2-Bromoethoxy)-2-nitrobenzene (66) 5 (20 g, 62.8 mmol) was dissolved in EtOH (200 mL). Pd/C (2 g) was O O
	NO 2		2-Nitrophenol (100 g, 719 mmol) was solubilized in hot MeOH and a mixture added and the flask placed under hydrogen atmosphere and stirred for
	of KOH (40 g, 719 mmol) in MeOH was added and a red precipitate was 24 h. The reaction mixture was filtered over celite, washed with hot EtOH and the solvent was O Br
	formed. The solvent was evaporated and the solid was dissolved in a minimum evaporated. The crude product was taken up in Et 2 O, sonicated and filtered. The precipitate
	amount of DMF (circa 200 mL). 1,2-dibromoethane (247 ml, 2.88 mol) is added and the was washed with cold Et 2 O and dried under vacuum to yield 23.8 g (91.1 mmol, 71 %) of
	reaction mixture heated to 120 °C for 4 h. After cooling, the reaction mixture was filtered and product.
	the filtrate washed with DCM and a small volume of water. The organic phase was washed 1 H NMR (300 MHz, CDCl 3 ): δ = 6.84 -6.63 (m, 7H), 4.35 (s, 4H), 3.73 (s, br, 4H), 2.26 (s,
	with NaOH (10 %, 3x) and brine (3x). The organic phase was dried over MgSO 4 , filtered and 3H). Spectra conform with literature
	evaporated. The crude product was recrystallized from hot EtOH containing a few drops of
	water to obtain 64.5 g (263 mmol, 36.5 %) of the product as a beige solid.
	Methyl-BAPTA (69) 5
	1 H NMR (300 MHz, CDCl 3 ): δ = 7.79 -7.06 (m, 8H), 4.41 (t, J = 7.02 Hz, 4H), 3.67 (t, J = 2-(2-(2-aminophenoxy)ethoxy)-4-methylaniline (22 g, 85.2 mmol)
	7.04 Hz, 2H). O O O	O	O	O	was dissolved in dry AcCN (150 ml), NaI (5.02 g, 33.2 mmol),
	Spectra conform with literature N N Na 2 HPO 4 (60.5 g, 425.9 mmol) and ethyl bromoacetate (52 ml, O O
	O	O				468.6 mmol) were added and the reaction mixture was heated at
						reflux overnight. After cooling to room temperature the solution
	4-methyl-1-nitro-2-(2-(2-nitrophenoxy)ethoxy)-benzene (67) 5

1

  NMR (75 MHz, CDCl 3 ): δ = 171.33, 171.23, 155.28, 150.79, 150.28, 143.10, 140.12, 137.10, 132.24, 128.04, 122.26, 121.13, 121.02, 119.76, 119.08, 115.40, 113.22, 67.55, 67.23, 60.84, 60.52, 53.67, 20.86, 14.53, 14.13, 14.09. HRMS (ESI): Calc. m/z = 871.3871 for C 44 H 55 BF 2 N 4 O 10 Na Found m/z = 871.3871 [M+Na] +

	1 H NMR (300 MHz, CDCl 3 ): δ = 6.87 (d, J = 8.5 Hz, 1H), 7.79-7.74 (m, 3H), 6.67 (d, J =
	7.2Hz, 2H), 5.97 (s, 2H), 4.25 (m, 4H), 4.21 (s, 4H), 4.10 (q, J = 7.1 Hz, 4H), 4.09 (s, 4H),
	4.07 (q, J = 7.1 Hz, 2H), 2.54 (s, 6H), 2.24 (s, 3H), 1.48 (s, 6H), 1.19 (t, J = 7.1 Hz, 6H), 1.16
	(t, J = 7.1 Hz, 6H).

13

C

  H NMR (300 MHz, CDCl 3 ): δ = 7.38 (dd, J = 1.7 Hz, J = 5.8 Hz, 1H), 6.88 -6.82 (m, 1H),

	6.77 -6.72 (m, 2H), 6.67 -6.58 (m, 2H), 4.33 -4.26 (m, 4H), 4.23 (s, 4H), 4.11 -4.00 (m,
	12H), 2.25 (s, 3H), 1.18 -1.12 (m, 12H). Spectra conform with literature
	BAPTA boronic ester (74)

1

  H NMR (300 MHz, CDCl 3 ): δ = 7.67 (d, J = 8.2 Hz, 2H), 7.30 (d, J = 7.9 Hz, 2H), 7.23 -7.19 (m, 2H), 6.90 (d, J = 8.2 Hz, 1H), 6.77 (d, J = 7.8 Hz, 1H), 6.70 -6.67 (m, 2H), 5.99 (s, 2H), 4.40 (m, 2H), 4.32 (m, 2H), 4.21 (s, 4H), 4.13 (s, 4H), 4.10 (q, J = 7.1 Hz, 4H), 4.06 (q, J = 7.1 Hz, 2H), 2.56 (s, 6H), 2.26 (s, 3H), 1.45 (s, 6H), 1.19 (t, J = 7.1 Hz, 6H), 1.16 (t, J = 7.1

	BAPTA-PH-BODIPY (76)
									The BAPTA-PH-BODIPY-ester (8 mg, 9 µmol) was dissolved in a
	O	O	O	O	O	O	O	O	mixture of THF (1 mL) and MeOH (1 mL), then LiOH.H 2 O (5 mg,
		N		O	O		N		120 µmol) and a drop of water was added and the mixture was
									stirred at room temperature overnight. The solvent was evaporated
									under reduced pressure and the crude product was dispersed in ethyl
									acetate and centrifuged. The solvent was pipetted off, the solid was
		N B	N						dissolved in water and acidified with 1M HCl. The precipitate was
	F F dissolved in an appropriate amount of KOH solution to form the potassium salt and freeze
	dried to yield the title compound quasi quantitatively.
	HRMS (FD):					Calc. m/z = 812.3040 for C 42 H 43 BF 2 N 4 O 10
									Found m/z = 812.3041 [M] +
	BANIF ester (78)
	Hz, 6H);						BAPTA-aldehyde (55 mg, 0.087 mmol) and C 8 F 17 -amino-
	13 C NMR (75 MHz, CDCl 3 ): δ = 171.59, 171.50, 171.37, 171.33, 155.41, 150.53, 150.28, naphthalimide (52 mg, 0.079 mmol) were dissolved in hexafluoro
	149.25, 143.13, 142.05, 141.65, 141.19, 139.39, 136.95, 136.88, 133.74, 133.34, 132.13,
	131.96, 131.48, 128.72, 128.68, 128.41, 127.01, 126.67, 121.95, 121.72, 121.17, 121.13,
	120.15, 119.46, 119.27, 119.18, 118.66, 117.55, 114.72, 114.35, 112.42, 83.51, 67.53, 67.08,
	66.98, 60.80, 60.60, 53.62, 24.83, 20.92, 14.60, 14.09.
	HRMS (ESI): Calc. m/z = 947.4184 for C 50 H 59 BF 2 N 4 O 10 Na
									Found m/z = 947.4183 [M+Na] +

1

  CDCl 3 ): δ = 171.66, 171.48, 164.56, 163.81, 150.88, 150.47, 139.63, 137.01, 125.06, 122.88, 120.50, 119.36, 114.92, 60.97, 60.74, 53.78, 21.05, 14.25. 19 F NMR (400 MHz, CDCl 3 ): δ = = -81.71 (t, J = 10.2 Hz, 3F), -115.26 --115.42 (m. 2F), -

	O O OH C NMR (75 MHz, 122.59 (s, br, 2F), -122.83 (s, br, 4F), -123.64 (s, br, 2F), -124.37 (s, br, 2F), -127.03 (s, br, O N N OH O HO O OH O 13
	2F).
	N	B	N
	F F
	O	O	O	O	O	O
			O	O
		N			N
			O	O
			NH
		O	N	O
				C 8 F 17
	1 H NMR (300 MHz, CDCl 3 ): δ = 8.59 (d, J = 8.5 Hz, 1H), 8.45 (d, J = 8.3 Hz, 1H), 8.14 (d, J
	= 8.3 Hz, 1H), 7.61 (t, J = 7.3 Hz, 1H), 6.97 -6.92 (m, 2H), 6.83 (d, J = 7.2 Hz, 1H), 6.78 -
						202

6.75 (m, 2H), 6.68 -6.65 (m, 2H), 5.70 (m, 1H), 4.54 -4.59 (m, 4H), 4.30 -4.26 (m, 4H), 4.16 -3.99 (m, 16H), 2.66 -2.49 (m, 2H), 2.24 (s, 3H), 1.20 -1.12 (m, 12H).

  . m/z = 1312.0 for C 46 H 34 F 17 N 4 O 12 K 4 EtAc (80/20, v/v). The product was further purified by recrystallisation from hot EtOH, yielding 232 mg (0.26 mmol, 47 % yield). CDCl 3 ): δ = 171.54, 171.46, 153.72, 150.90, 150.45, 140.13, 138.55, 137.25, 132.77, 132.39, 131.13, 129.10, 122.37, 121.57, 119.89, 77.37, 61.01, 60.70, 53.83, 21.05, 17.22, 14.76, 14.28, 12.01. 19 F NMR (400 MHz, CDCl 3 ): δ = -146.82 (dd, J = 30.59 Hz, J = 65.61 Hz, 2F). , 108.47, 67.61, 67.32, 61.01, 60.66, 55.43, 53.82, 34.07, 33.28, 29.85, 21.01, 17.24, 14.63, 14.25, 12.56, 12.09.) was added dropwise via a syringe pump. The reaction was stirred for 2 h and subsequently a saturated solution of NaHCO 3 was added, the phases were separated and the aqueous phases were extracted with EtAc. The combined organic phases were dried over MgSO 4 , filtered and evaporated. The crude product was dissolved in THF (10 mL) and added dropwise to a solution of LiAlH 4 (994 mg, 26.8 mmol) in THF (130 mL)

	110.9219 F NMR (400 MHz, CDCl 3 ): δ = -81.73 (t, J = 9.71 Hz, 6F), -114.30 --114.77 (m, 4F), -1 H NMR (300 MHz, DMSO): δ = 7.63 (d, J = 9.0 Hz, 1H), 7.27 (d, J = 2.6 Hz, 1H), 7.12 (dd, J = 9.0 Hz, J = 2.6 Hz, 1H), 6.85 (s, 1H), 4.35 (q, J = 7.2 Hz, 2H), 3.80 (s, 3H), 1.33 (t, J = 7.2 Hz, 3H). (5-(Benzyloxy)benzofuran-2-yl)methanol (86) 2-(Hydroxymethyl)benzofuran-5-ol (1.9 g, 11.6 mmol) and benzyl bromide (1.5 ml, 12.7 mmol) were dissolved in AcCN (70 mL) and O 5-(benzyloxy)-6-nitrobenzofuran-2-carbaldehyde (88) PCC (2.9 g, 13.4 mmol) was dissolved in DCM (20 mL) and a solution of (5-(benzyloxy)-6-nitrobenzofuran-2-yl)methanol (2.0 g, cyclohexane/1 H NMR (400 MHz, CDCl 3 ): δ = 6.88 (d, J = 8.57 Hz, 1H), 6.78 -6.76 (m, 3H), 6.69 -6.67 122.68 (s, 4F), -122.89 (s, 8F), -123.68 (s, 4F), -124.65 (s, 2F), -124.87 (s, 2F), -127.07 (s, 13 C NMR (75 MHz, CDCl 3 ): δ = 159.65, 156.64, 150.95 146.44, 127.58, 117.59, 113.91, K 2 CO 3 (3.0 g, 21.7 mmol) was added. The reaction mixture was O OH 6.6 mmol) in DCM (60 mL) was added in one shot. The reaction
	(m, 2H), 4.28 -4.23 (m, 4H), 4.22 (s, 4H), 4.15 -4.03 (m, 12H), 2.52 (s, 6H), 2.30 (q, J = 4F). 113.08, 103.87, 61.58, 55.95, 14.46 heated to reflux for 5 h, then subsequently cooled to room temperature mixture was stirred at room temperature for 4 h and subsequently
	7.48 Hz, 4H), 2.24 (s, 3H), 1.39 (s, 6H), 1.22 -1.16 (m, 12H), 0.98 (t, J = 7.50 Hz, 6H). HRMS (FD): Calc. m/z = 1815.4628 for C 68 H 71 BN 4 O 12 F 34 Na HRMS (EI): Calc. m/z = 220.0713 for C 12 H 12 O 4 and filtered. The crude product was purified by column chromatography using PTE/EtAc washed with a saturated solution of NaCl (2x) NaHCO 3 (2x) and water. The organic phase
	Found m/z = 1312.5 [M] + (Potassium salt) OH Found m/z = 1815.4510 [M+Na] + C 8 F 17 -BAPTA-BODIPY (82) O O N N OH O O OH HO O O NH N O O C 8 F 17 13 Found m/z = 220.7356 [M] + 2-(Hydroxymethyl)benzofuran-5-ol (85) (60/40, v/v) to yield 2.67 g (10.5 mmol, 95 %) of product. 1 H NMR (300 MHz, CDCl 3 ): δ = 7.47 -7.33 (m, 6H), 7.09 (d, J = 2.6 Hz, 1H), 6.97 (dd, J = 8.9 Hz, J = 2.4 Hz, 1H), 6.59 (s, 1H), 5.09 (s, 1H), 4.75 (s, 1H), 1.31 (s, br, 1H). was dried over MgSO 4 , filtered and evaporated. The crude product was purified by column chromatography using PTE/EtAc (60/40, v/v) to yield 1.3 g (4.37 mmol, 66 %) of the aldehyde. C NMR (75 MHz, HRMS (FD): Calc. m/z = 927.4498 for C 48 H 63 BN 4 O 10 F 2 Na Found m/z = 927.4497 [M+Na] + C 8 F 17 -BAPTA-BODIPY ester (81) BAPTA-BODIPY2 (50 mg, 0.055 mmol) was dissolved in DCM (5 ml) and AlCl 3 (11 mg, 0.082 mmol) was added portion wise. The ethanol (56 mg, 0.121 mmol) was added in one portion and stirring was continued for 1 h. The reaction was cooled in an ice bath and sat. Sodium bicarbonate solution was added until CO 2 development stopped. The aqueous phase was extracted with DCM (3x) and the organic phase was washed with brine (2x). The aqueous phase was O O N B F 17 C 8 C 8 F 17 MS (MALDI) 1H), 6.45 (s, 1H), 4.59 (s, 2H), 3.46 (s, 3H). saturated NaCl solution and then dried over MgSO 4 . The solvent was evaporated after Found m/z = 1704.9 [M] + (Lithium salt) 4 Hz, and the aqueous phase was extracted with DCM (3x) The organic phase was washed with Calc. m/z = 1704.4 for C 60 H 51 BF 34 N 4 O 12 Li 4 O O syringe pump. After the completion of the reaction, water was added N chromatography using PTE/EtAc (50/50, v/v) to yield 2.0 g (12.1 mmol, 48 %) of product. O OH O 2 N (1.06 mL, 25.2 mmol) in DCM (30 mL) was added dropwise via a quantitatively. over MgSO 4 and evaporated after filtration. The crude product was purified by column O dissolved in DCM (60 mL) and cooled to -20 °C. Fuming HNO 3 solution to form the potassium salt and freeze dried to yield the title compound quasi-reaction was stirred for 20 min at room temperature, then C 8 F 17 -N N O O O O O O O O The C 8 F 17 -BAPTA-BODIPY-ester (20 mg, 11 µmol) was dissolved in a mixture of THF (1 mL) and MeOH (1 mL), then O O N N OH O OH HO O OH O Ethyl 5-methoxybenzofuran-2-carboxylate (5.7 g, 25.9 mmol) was dissolved 13 C NMR (75 MHz, CDCl 3 ): δ = 157.41, 155.20, 137.3, 128.66, 127.59, 114.02, 111.80, 1 H NMR (300 MHz, CDCl 3 ): δ = 9.91 (s, 1H), 8.07 (s, 1H), 7.50 -7.36 (m, 6H), 5.27 (s, 1H). HO O in DCM (70 mL) and cooled to -60 °C. A 1 M solution of BBr 3 in DCM O 105.26, 104.42, 71.00, 58.27. 13 C NMR (75 MHz, CDCl 3 ): δ = 179.70, 156.04, 148.96, 141.62, 135.39, 130.65, 128.93, OH LiOH.H 2 O (10 mg, 250 µmol) and a drop of water was added and the mixture was stirred at room temperature overnight. The solvent was evaporated under reduced pressure and the crude product was dispersed in ethyl acetate and centrifuged. The solvent was pipetted off, the solid was dissolved in water and acidified with 1M HCl. The precipitate was dissolved in an appropriate amount of KOH N B N O O F 17 C 8 C8F17 HRMS (ESI): Calc. m/z = 255.0943 for C 16 H 15 O 3 (5-(benzyloxy)-6-nitrobenzofuran-2-yl)methanol (87) (5-(Benzyloxy)benzofuran-2-yl)methanol (2.7g, 10.5 mmol) were 128.51, 127.17, 115.47, 110.23, 108.21, 72.15. (53 mLand stirred for 30 min. A saturated NH 4 Cl solution was added and extracted with EtAc, dried Found m/z = 255.0948 [M+H] + HRMS (ESI): Calc. m/z = 296.0570 for C 16 H 10 NO 5
	BAPTA-BODIPY2 ester (80) dried over MgSO 4 , filtered and evaporated to dryness. The crude product was purified by 13 C NMR (75 MHz, MeOD): δ = 159.80, 154.60, 130.93, 114.23, 112.47, 107.08, 105.01, filtration and the crude product was purified by column chromatography using PTE/EtAc
	In a round bottom flask, BAPTA-aldehyde (345 mg, 0.55 mmol) and kryptopyrrole (0.22 mL, 1.64 mmol) were dissolved in freshly-distilled DCM (80 mL) and a drop of TFA was added. The reaction mixture was stirred at room temperature until the aldehyde was consumed. The p-chloranil (135 mg, 0.55 mmol) column chromatography using PTE/EtAc (90/10, v/v) to obtain 25 mg (0.014 mmol, 26 % O O N N O O O O O O O O N B N (50/50, v/v) to yield 2.0 g (6.69 mmol, 63 %) of product. 58.48. Ethyl 5-methoxybenzofuran-2-carboxylate (84) yield) of the product as an amorphous orange solid. HRMS (ESI): Calc. m/z = 163.0399 for C 9 H 7 O 3 5-Methoxysalicylaldehyde (5.5 g, 36.2 mmol) and ethyl bromo-acetate O O 1 H NMR (300 MHz, CDCl 3 ): δ = 8.02 (s, 1H), 7.51 -7.50 (m, 2H), 7.43 -7.33 (m, 3H), 7.20 Found m/z = 163.0400 [M-H] -(4.7 ml, 43.1 mmol) were dissolved in AcCN (200 mL) and K 2 CO 3 (14.9 g, O O (s, 1H), 6.67 (s, 1H), 5.25 (s, 2H), 4.79 (s, 2H), 1.99 (s, br, 1H). 1 H NMR (400 MHz, DMSO-d 6 ): δ = 6.89 (d, J = 7.90 Hz, 1H), 6.79 -6.74 (m, 3H), 6.69 -6.68 (m, 2H), 4.27 (s, br, 4H), 4.23 (s, 4H), 4.13 -4.05 (m, 12H), 4.00 (dd, J = 6.02 Hz, J = 108 mmol) was added. The reaction mixture was stirred under reflux 13 C NMR (75 MHz, CDCl 3 ): δ = 162.49, 149.09, 147.78, 135.97, 133.40, 128.81, 128.31, overnight, then cooled to room temperature and filtered. The solvent was evaporated and the in DCM (20 mL) was added and stirring was continued for F F 11.95 Hz, 4H), 3.35 -3.26 (m, 4H), 2.51 (s, 6H), 2.31 (q, J = 6.87 Hz, 4H), 2.25 (s, 3H), 1.40 127.22, 109.15, 106.73, 104.11, 72.29, 58.19. crude product was purified by column chromatography using PTE/EtAc (90/10, v/v) to yield 30 min. Borontrifluoride diethyl etherate (3.45 mL) and TEA HRMS (ESI): Calc. m/z = 322.0690 for C 16 H 13 NO 5 Na 3.5 g (15.9 mmol, 44 %) of the product as a white solid. Found m/z = 322.0685 [M+Na] +
	204

(3.45 mL) were added and stirring was continued for 2 h. H 2 O was added and the aqueous phase was extracted with DCM (3x) and dried over MgSO 4 . After filtration, the solvent was evaporated and the crude product was purified by column chromatography using (s, 6H), 1.

21

-1.16 (m, 12H), 0.98 (t, J = 7.44 Hz, 6H). 13 C NMR (75 MHz, CDCl 3 ): δ = 171.54, 153.98, 150.93, 150.45, 140.38, 140.08, 137.60, 137.26, 133.01, 132.41, 132.35, 129.34, 122.37, 121.72, 119.91, 119.28, 115.48, 113.49,

1 H NMR (300 MHz, MeOD): δ = 7.18 (d, J = 8.5 Hz, 1H), 6.70 (dd, J = 9.8 Hz, J = 2.

  The reaction was cooled, a saturated solution of NaHCO 3 was added and the aqueous phase was extracted with DCM. The combined organic phases were dried over MgSO 4 , filtered and evaporated and the crude product was purified by column chromatography using PTE/DCM (50/50, v/v) to yield 570 mg (2.15 mmol, 69 %) of product. The reaction mixture was heated to 120 °C for 1 h, then cooled to room temperature and brine was added. The aqueous phase was extracted with EtAc (3x) and the combined organic phases were washed with brine (2x) and water. The organic phase was dried over MgSO 4 , filtered and evaporated. The crude was dissolved in a minimum amount of acetone and under stirring PTE was added. The product precipitated from the solution to yield 0.74 g (1.37 mmol, 75 %) of pure product. The reaction was filtered over celite and washed with EtOH. The solvent was evaporated and the crude was purified by column chromatography using DCM/EtAc (90/10, v/v) to yield 130 mg (0.27 mmol, 73 %) of the diamine. HO-BAPTA acetal (110 mg, 0.15 mmol) and C 8 F 17 -butyl bromide (91 mg, 0.16 mmol) were dissolved in AcCN (5 mL). K 2 CO 3

	O 2-(1,3-Dioxan-2-yl)-6-nitrobenzofuran-5-ol (90) H O O 2 N O HRMS (FD): Calc. m/z = 355.1062 for C 19 H 17 NO 6 Found m/z = 355.1056 [M] + 13 C NMR (75 MHz, CDCl 3 ): δ = 153.14, 145.91, 136.00, 128.87, 128.50, 127.66, 121.62, 118.36, 111.28, 71.12, 28.56. HRMS (FD): Calc. m/z = 351.0095 for C 15 H 14 NO 4 Br Found m/z = 351.01062 [M] + HRMS (FD): Calc. m/z = 820.3456 for C 43 H 52 N 2 O 14 stirred overnight. 1 H NMR (300 MHz, CDCl 3 ): δ = 7.43 -7.31 (m, 5H), 6.96 (s, 1H), 6.85 (s, 1H), 6.77 (d, J = Found m/z = 820.3419 [M] +
	5-(Benzyloxy)-2-(1,3-dioxan-2-yl)-6-nitrobenzofuran (1.1 g, 3.1 mmol) 8.7 Hz, 1H), 6.65 (s, 1H), 6.40 (d, J = 3.0 Hz, 1H), 6.31 (dd, J = 8.9 Hz, J = 2.8 Hz, 1H), 5.65 HO O HO-BAPTA-acetal (94)
	was dissolved in CHCl 3 (85 mL) and cooled to 0 °C. AlCl 3 (0.8 g, 5-(2-(4-(benzyloxy)-2-nitrophenoxy)ethoxy)-2-(1,3-dioxan-2-yl)-6-nitrobenzofuran (91) O O O 2 N (s, 1H), 4.98 (s, 2H), 4.34 -4.27 (m, 6H), 4.03 -3.95 (m, 2H), 2.34 -2.22 (m, 1H), 1.50 -Bz-BAPTA-acetal (40 mg, 0.05 mmol) was dissolved in EtAc
	NO 2 1.45 (m, 1H).	NO 2	6.1 mmol) was added in small portions and the reaction mixture was 2-(1,3-Dioxan-2-yl)-6-nitrobenzofuran-5-ol (0.50 g, 1.88 mmol) and (2 mL) and Pd/C (10 mg) was added. The reaction mixture was
	4-(benzyloxy)-1-(2-bromoethoxy)-2-nitrobenzene 1.82 mmol) were dissolved in DMF and K 2 CO 3 (0.13 g, 0.94 mmol) (0.64 g, C NMR (75 MHz, CDCl 3 ): δ = 154.44, 151.13, 150.95, 144.19, 140.90, 138.33, 137.55, O O O O O 13 136.04, 128.63, 127.91, 127.54, 118.19, 114.39, 104.54, 104.39, 103.52, 103.17, 97.77, 96.52, placed under a hydrogen atmosphere, stirred overnight and subsequently filtered over celite and washed with EtAc. The heated to 60 °C for 1 h. HRMS (ESI): Calc. m/z = 264.0517 for C 12 H 10 NO 6 O 70.49, 68.69, 68.45, 67.44, 25.79. HRMS (FD): Calc. m/z = 477.1981 for C 25 H 30 N 2 O 6 Na Found m/z = 477.1996 [M+Na] + Bz-BAPTA-acetal (93) 5-(2-(2-amino-4-(benzyloxy)phenoxy)ethoxy)-2-(1,3-dioxan-2-solvent was evaporated and the crude product was purified by column chromatography using PTE/EtAc (50/50, v/v) to yield 31 mg (0.04 mmol, 88 %) of product. was added. 1 H NMR (300 MHz, CDCl 3 ): δ = 8.00 (s, 1H), 7.45 -7.44 (m, 1H), 7.41 -7.34 (m, 6H), 7.19 yl)benzofuran-6-amine (150 mg, 0.31 mmol), ethyl bromoacetate O O O O O O O O 13 C NMR (75 MHz, CDCl 3 ): δ = 171.61, 171.56, 152.48, 150.49, 150.29, 148.32, 144.51,
	Found m/z = 264.0513 [M-H] -(d, J = 1.6 Hz, 2H), 6.87 (s, 1H), 5.71 (s, 1H), 5.06 (s, 2H), 4.52 -4.46 (m, 4H), 4.30 (dd, J = N N (348 µL, 3.1 mmol) and diisopropylethylamine (DIPEA) (263 µL, O O 140.77, 138.49, 121.33, 115.15, 107.81, 106.79, 104.80, 104.39, 102.57, 96.41, 68.08, 67.48,
	10.8 Hz, J = 4.7 Hz, 2H), 4.02 (dt, J = 12.4 Hz, J = 2.6 Hz, 2H), 2.36 -2.20 (m, 1H), 1.54 -1.55 mmol) were dissolved in NMP (6 mL) and heated to 120°C O O 60.98, 53.92, 25.77, 14.24, 14.20.
	O 4-(benzyloxy)-1-(2-bromoethoxy)-2-nitrobenzene (90-1) O O O 2 N O 1.48 (m, 1H). 13 C NMR (75 MHz, CDCl 3 ): δ = 158.81, 153.00, 149.15, 147.89, 146.56, 140.73, 138.35, for 3 h. The reaction mixture was cooled to room temperature and O HRMS (FD): Calc. m/z = 730.2924 for C 36 H 46 N 2 O 14 O diluted with EtAc. The organic phase was washed with brine (3x) Found m/z = 730.2949 [M] +
	4-(Benzyloxy)-2-nitrophenol (1.0 g, 3.5 mmol) was dissolved in AcCN (40 mL) and K 2 CO 3 (1.4 g, 10.6 mmol) was added. The mixture was and water (3x) and dried over MgSO 4 . After filtration and evaporation of all volatiles, the 136.10, 132.85, 128.84, 128.45, 127.68, 121.79, 118.53, 111.27, 109.35, 108.31, 104.64, NO 2 O Br 95.80, 71.11, 70.15, 67.49, 25.70. crude was purified by column chromatography using PTE/EtAc (60/40, v/v) to yield 130 mg
	heated to 60 °C and a solution of 1.2-dibromoethane (3.0 ml, 35 mmol) in Calc. m/z = 559.1344 for C 27 H 24 N 2 O 10 Na (0.16 mmol, 51 %) of product. O HRMS (FD): C 8 F 17 -BAPTA-acetal (95)
	AcCN (50 mL) was added dropwise. The reaction mixture was refluxed for 3 h, then cooled and filtered. The solid was washed with DCM and the combined filtrates were evaporated. Found m/z = 559.1323 [M+Na] + 1 H NMR (300 MHz, CDCl 3 ): δ = 7.42 -7.30 (m, 5H), 6.99 (d, J = 8.7 Hz, 2H), 6.77 (d, J =
	1 H NMR (300 MHz, CDCl 3 ): δ = 8.03 (s, 1H), 7.49 -7.46 (m, 2H), 7.42 -7.32 (m, 3H), 7.23 The crude product was purified by column chromatography using PTE/DCM (60/40, v/v) to 9.0 Hz, 1H), 6.66 (s, 1H), 6.48 -6.45 (m, 2H), 5.66 (s, 1H), 4.97 (s, 2H), 4.32 -4.24 (m, 6H),
	(s, 1H), 6.80 (s, 1H), 5.70 (s, 1H), 5.24 (s, 2H), 4.30 (dd, J = 10.8 Hz, J = 5.2 Hz, 2H), 4.02 yield 0.78 g (2.22 mmol, 64 %) of product. 5-(2-(2-amino-4-(benzyloxy)phenoxy)ethoxy)-2-(1,3-dioxan-2-yl)benzofuran-6-amine (92) 4.17 (s, 4H), 4.14 (s, 4H), 4.10 -3.96 (m, 10H), 2.36 -2.20 (m, 1H), 1.50 -1.45 (m, 1H),
	(dt, J = 12.1 Hz, J = 2.6 Hz, 2H), 2.36 -2.202 (m, 1H), 1.55 -1.47 (m, 1H). 5-(2-(4-(benzyloxy)-2-nitrophenoxy)ethoxy)-2-(1,3-dioxan-2-yl)-6-NH 2 NH 2 1.18 -1.10 (m, 12H).
	O C NMR (75 MHz, CDCl 3 ): δ = 171.50, 171.46, 153.78, 152.51, 150.31, 148.34, 144.91, O 13
	O		O
	O
	O
			209

13

C NMR (75 MHz, CDCl 3 ): δ = 158.

72, 149.01, 138.30, 135.94, 132.58, 128.78, 127.22, 109.49, 107.12, 104.42, 95.78, 72.25, 7.48, 25.

69. 1 H NMR (300 MHz, CDCl 3 ): δ = 10.48 (s, 1H), 8.23 (s, 1H), 7.23 (s, 1H), 6.78 (s, 1H), 5.69 (s, 1H), 4.35 -4.26 (m, 2H), 4.08 -3.95 (m, 2H), 2.40 -2.16 (s, 1H), 1.55 -1.48 (s, 1H). 13 C NMR (75 MHz, CDCl 3 ): δ = 160.80, 151.54, 147.75, 137.05, 131.44, 109.79, 17.81, 104.25, 95.66, 67.50, 25.68.

1 H NMR (300 MHz, CDCl 3 ): δ = 7.46 (d, J = 3.0 Hz, 1H), 7.42 -7.32 (m, 5H), 7.16 (dd, J = 9.0 Hz, J = 3.0 Hz, 1H), 7.06 (d, J = 9.4 Hz, 1H), 5.07 (s, 2H), 4.36 (t, J = 6.6 Hz, 2H), 3.65 (t, J = 6.6 Hz, 2H). nitrobenzofuran (200 mg, 0.37 mmol) was dissolved in a mixture of EtOH and EtAc (1:1, v/v) and Pt/C (20 mg) was added. The reaction mixture was placed under a hydrogen atmosphere and 140.75, 138.

53, 137.39, 128.64, 127.99, 127.63, 121.35, 107.40, 106.61, 104.94, 104.36, 102.61, 96.40 70.60, 68.03, 67.45, 60.91, 53.93, 53.62, 25.78, 14.20, 14.17

.

1 H NMR (300 MHz, CDCl 3 ): δ = 6.99 (d, J = 18.0 Hz, 2H), 6.71 (d, J = 6.3 Hz, 1H), 6.66 (s, 1H), 6.38 -6.32 (m, 2H), 5.66 (s, 1H), 4.30 -4.21 (m, 6H), 4.17 (s, 4H), 4.14 (s, 4H), 4.11 -3.98 (m, 10H), 2.32 -2.24 (m, 1H), 1.49 -1.46 (m, 1H), 1.21 -1.16 (m, 12H).

  1 H NMR (400 MHz, CDCl 3 ): δ = 6.99 (d, J = 10.7 Hz, 2H), 6.77 (d, J = 8.5 Hz, 1H), 6.66 (s, 1H), 6.41 -6.38 (m, 2H), 5.65 (s, 1H), 4.30 -4.23 (m, 6H), 4.17 (s, 4H), 4.14 (s, 4H), 4.09 -4.00 (m, 10H), 3.90(t, J = 5.4 Hz, 2H), 2.34 -2.23 (m, 1H), 2.21 -2.08 (m, 2H), 1.81 -1.76 (m, 4H), 1.49 -1.46 (m, 1H), 1.20 -1.11 (m, 12H). 13 C NMR (75 MHz, CDCl 3 ): δ = 171.63, 171.59, 153.92, 152.59, 150.40, 148.41, 144.95, 140.87, 138.59, 121.45, 114.97, 107.17, 106.30, 105.04, 104.45, 102.70, 96.48, 68.09, 67.80, 67.55, 61.00, 54.02, 53.72, 30.92, 28.99, 25.84, 17.48, 14.27, 14.23. 19 F NMR (400 MHz, CDCl 3 ): δ = = -81.74 (t, J = 10.2 Hz, 3F), -115.50 --115.67 (m. 2F), -122.90 (s, br, 2F), -123.09 (s, br, 4F), -123.88 (s, br, 2F), -124.65 (s, br, 2F), -127.27 (s, br, 2F). HRMS (FD): Calc. m/z = 1204.3196 for C 48 H 53 F 17 N 2 O 14 Found m/z = 1204.3225 [M] + C 8 F 17 -fura red ester (96) C 8 F 17 -BAPTA-acetal (50 mg, 0.04 mmol) and 2-thiohydantoin (5.3 mg, 0.05 mmol) were dissolved in AcOH (2.5 mL) along with NH 4 OAc (11 mg, 0.14 mmol). The reaction mixture was stirred at 60 °C for 2 h, cooled and water was added. The aqueous phase was extracted with EtAc (3x) and the combined organic phases washed with a saturated solution of NaHCO 3 . The organic phase was dried over Na 2 SO 4 , filtered and evaporated. The crude product was purified by column chromatography using DCM/EtAc (90/10, v/v) to yield 40 mg (0.03 mmol, 80 %) of product. CDCl 3 ): δ = 175.08, 171.52, 171.32 163.60, 154.00, 152.29, 150.65, 148.97, 144.77, 141.13, 140.84, 125.73, 121.80, 115.15, 113.51, 107.24, 106.32, 104.43, 101.60, 100.72, 67.99, 67.73, 61.37, 60.94, 54.07, 53.64, 30.84, 28.92, 17.42, 14.24, 14.19, 19 F NMR (400 MHz, CDCl 3 ): δ = = -81.94 (t, J = 9.5 Hz, 3F), -115.55 --115.62 (m. 2F), -122.90 (s, br, 2F), -123.09 (s, br, 4F), -123.88 (s, br, 2F), -124.65 (s, br, 2F), -127.27 (s, br, 2F). HRMS (FD): Calc. m/z = 1244.2774 for C 48 H 49 F 17 N 4 O 13 S Found m/z = 1244.2745 [M] + C 8 F 17 -fura red ester (97) C 8 F 17-furared-ester (15 mg, 12 µmol) was dissolved in a mixture of THF (1 mL) and MeOH (1 mL), then LiOH.H 2 O (10 mg,

	13 C NMR (75 MHz,
	O	O	O	O O	O
			O	O
		N		N
			O	O
	O				O
			O
	HN	NH	C 8 F 17
	S
	1 H NMR (400 MHz, CDCl 3 ): δ = 9.61 (s, 1H), 9.96 (s, 1H), 7.12 (s, 1H), 6.99 (s, 1H), 6.91 (s,
	1H), 6.78 (d, J = 8.6 Hz, 1H) 6.50 (s, 1H), 6.42 (d, J = 9.3 Hz, 2H), 4.27 -4.23 (m, 8H), 4.17
	(s, 4H), 4.13 -4.03 (m, 8H), 3.90 (t, , J = 5.7 Hz, 2H), 2.19 -2.09 (m, 2H), 1.85 -1.78 (m,

4H), 1.21 -1.14 (m, 12H).

  96, 171.41, 153.97, 147.99, 140.50, 132.44, 109.87, 107.93, 78.35, 70.52, 70.46, 68.97, 61.34, 60.53, 60.45, 56.72, 56.46, 55.98, 55.82, 53.74, 14.39, 14.35. HRMS (ESI): Calc. m/z = 696.2952 for C 30 H 47 N 3 O 14 Na Found m/z = 696.2950 [M+Na] +

  J = 2.63, J = 4.40, 1H), 3.97 (s, 3H) 4.29 (dd, J = 4.46, J = 5.53, 1H), 2.64 (dd, J = 2.59, J = 5.55, 1H).13 C NMR (75 MHz, CDCl 3 ): δ = 154.96, 147.27, 140.05, 135.76, 130.18, 128.86, 128.50, 127.68, 110.05, 108.36, 71.44, 56.63, 51.35, 50.68.HRMS (ESI): Calc. m/z = 302.1029 for C 16 H 16 NO 5 Found m/z = 302.1022 [M+H] + 2-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)-2-(2-(2-chloroethoxy)ethoxy)ethanol (116) In a round bottom flask, 2-(4-(benzyloxy)-5-methoxy-2nitrophenyl)oxirane (1.80 g, 5.97 mmol) was dispersed in 2-(2chloroethoxy)ethanol (6.9 ml, 66.0 mmol) and a minimum amount of THF was added to solubilise all solids. Eu(CF 3 SO 3 ) 3 (50.0 mg, 0.083 mmol) was added and the reaction mixture stirred at 50 °C for 2 h. Water was added and the aqueous phase extracted with DCM (3x) The organic phase was dried over MgSO 4 , filtered and evaporated to dryness. The crude product was purified by column chromatography starting with two column volumes PTE/EtAc (80/20, v/v) to wash of the excess of 2-(2-chloroethoxy)ethanol then eluting the rest with PTE/EtAc (50/50, v/v). 1.81 g (4.26 mmol, 71 % yield) of the product was obtained. H NMR (300 MHz, DMSO-d 6 ): δ = 7.71 (s, 1H), 7.50-7.31 (m, 5H), 7.24 (s, 1H), 5.19 (s, 2H), 5.03 (dd, J = 4.25, J = 6.01, 1H), 3.90 (s, 3H) 3.73-3.43 (m, 11H). = 153.37, 146.53, 140.63, 136.25, 130.43, 128.49, 128.11, 127.94, 110.19, 109.24, 78.66, 70.58, 70.33, 69.75, 68.61, 56.10, 43.49. HRMS (ESI): Calc. m/z = 448.1114 for C 20 H 24 NO 7 ClNa Found m/z = 448.1133 [M+Na] + (benzyloxy)-4-(1-(2-(2-chloroethoxy)ethoxy)-2-iodoethyl)-2-methoxy-5-nitrobenzene (117) In a round bottom flask, imidazole (0.74 g, 10.80 mmol) and 2-(4-(benzyloxy)-5-methoxy-2-nitrophenyl)-2-(2-(2-chloro-ethoxy) ethoxy)ethanol (1.60 g, 3.76 mmol) were dissolved in toluene (40 mL), then PPh 3 (2.10 g, 8.01 mmol) and I 2 (1.46 g, 5.76 mmol) were added. The reaction mixture was heated to 80 °C for 1.5 h. After cooling to room temperature, the solution was diluted with EtAc and washed with a saturated aqueous Na 2 SO 4 solution and then a saturated aqueous NaHCO 3 solution. The organic phase was dried over MgSO 4 , filtered and the solvent was evaporated. The crude product was then purified by column chromatography using first PTE/EtAc (90/10, v/v) then PTE/EtAc (70/30, v/v) to yield 1.40 g (2.62 mmol, 70 % yield) of pure product.1 H NMR (300 MHz, DMSO-d 6 ): δ = 7.76 (s, 1H), 7.49-7.35 (m, 5H), 7.29 (s, 1H), 5.21 (s, 2H), 5.09 (dd, J = 2.78, J = 8.43, 1H), 3.93 (s, 3H) 3.71-3.36 (m, 10H). 13 C NMR (75 MHz, DMSO): δ = 153.69, 146.94, 140.07, 136.13, 130.89, 128.50, 128.14, 127.95, 109.64, 109.38, 77.02, 70.56, 70.37, 69.64, 68.82, 56.23, 43.45. HRMS (ESI): Calc. m/z = 558.0162 for C 20 H 23 NO 6 IClNa Found m/z = 558.0150 [M+Na] +

	1-	O
	O O 4.52 (dd, O	H NO 2 O O	OH Cl
	O	NO 2
		O
	O
	O	NO 2

1 13 C NMR (75 MHz, DMSO): δ

  1 H NMR (300 MHz, CDCl 3 ): δ = 7.70 (s, 1H), 7.48-7.34 (m, 5H), 7.28 (s, 1H), 5.19 (s, 2H),5.07 (dd, J = 2.84, J = 7.77, 1H), 3.99 (s, 3H) 3.75-3.56 (m, 6H) 3.06 (dd, J = 2.86, J = 13.6, 1H) 2.88-2.76 (m, 3H).13C NMR (75 MHz, CDCl 3 ): δ = 154.51, 147.09, 140.62, 135.83, 132.43, 128.85, 128.47, 127.66, 110.13, 109.65, 80.73, 73.59, 71.37, 70.46, 69.20, 56.56, 48.76, 41.85. In a round bottom flask equipped with a reflux condenser, Na 2 HPO 4 (1.40 g, 9.86 mmol), NaI (0.15 g, 1.00 mmol), ethyl bromoacetate (2.2 mL, 19.7 mmol) and 2-(2-(2aminoethoxy)ethoxy)-2-(4-(benzyloxy)-5methoxy-2-nitrophenyl)ethanamine (0.80 g, 1.97 mmol) were dispersed in acetonitrile (25 mL). The reaction mixture was heated under reflux overnight. The solution was cooled to room temperature and filtered. The solid was filtered off and washed with acetonitril. The filtrate was evaporated to dryness and the crude product was purified by column chromatorgraphy using PTE/EtAc (30/70, v/v) to yield 1.13 g (1.51 mmol, 76 %) product as a light yellow oil. H NMR (300 MHz, CDCl 3 ): δ = 7.66 (s, 1H), 7.46-7.32 (m, 5H), 7.28 (s, 1H), 5.27 (dd, J = 2.29, J = 7.59, 1H), 5.17 (s, 2H), 4.18-4.09 (m, 8H), 3.98 (s, 3H), 3.75 (s, 4H), 3.57 (s, 4H), 3.57-3.37 (m, 6H), 3.08 (dd, J = 2.37, J = 14.70, 1H), 2.98 (dd, J = 8.28, J = 15.39, 1H), 2.92 (t, J = 5.79, 2H), 1.28-1.21 (m, 12H). NMR (75 MHz, CDCl 3 ): δ = 171.98, 171.43, 154.57, 146.98, 140.28, 135.94, 132.72, 128.86, 128.46, 127.67, 120.00, 110.08, 78.38, 71.39, 70.51, 70.47, 68.97, 61.35, 60.56, 60.47, 56.71, 55.99, 55.81, 53.74, 14.40, 14.36. HRMS (ESI): Calc. m/z = 772.3259 for C 36 H 51 N 3 O 14 Na Found m/z = 772.3263 [M+Na] +

	MS (ESI):	Calc. m/z = 405.190 for C 20 H 27 N 3 O 6
		Found m/z = 406.2 [M+H] +
	Bz-DMNPE-4 ester (119)

1 13 C

  H NMR (400 MHz, CDCl 3 ): δ = 7.58 (s, 1H), 7.24 (s, 1H), 5.27 (dd, J = 2.12, J = 7.76, 1H), 4.17-4.10 (m, 8H), 4.03 (t, J = 6.81, 2H), 3.96 (s, 3H), 3.75 (s, 4H), 3.57 (s, 4H), 3.56-3.38 (m, 6H), 3.08 (dd, J = 2.18, J = 14.69, 1H), 2.98 (dd, J = 7.80, J = 14.75, 1H), 2.92 (t, J = 5.77, 2H), 1.88-1.75 (m, 3H), 1.48-1.41 (m, 2H), 1.36-1.21 (m, 31H) 0.87 (t, J = 6.84, 3H). 13 C NMR (75 MHz, CDCl 3 ): δ = 172.00, 171.43, 154.31, 147.51, 140.40, 132.06, 109.84, 108.97, 78.31, 70.46, 69.58, 68.89, 61.32, 60.55, 60.48, 56.67, 55.96, 55.78, 53.72, 32.04, 29.78, 29.48, 29.02, 26.02, 22.81, 14.37. HRMS (ESI): Calc. m/z = 878.4994 for C 43 H 73 N 3 O 14 Na Found m/z = 878.4984 [M+Na] +

1

  H NMR (300 MHz, CDCl 3 ): δ = 4.53-4.62 (m, 1H), 3.90-3.97 (m, 1H), 3.80-3.87 (m, 1H), 2.81-3.13 (m, 2H) 1.99-2.17 (m, 2H), 1.53 (s, br, 1H). CDCl 3 ): δ = 62.55, 42.37, 42.34, 41.99, 16.64. 19 F NMR (400 MHz, CDCl 3 ): δ = -80.74 (s, br,, 3F), -110.52-114.97 (m, 2F), -121.55 (s, br, 2F), -121.85 (s, br, 4F), 122.68 (s, br, 2F), 123.56 (s, br, 2F), 126.06 (s, br, 2F).Heptadecafluoro-3-iododecan-1-ol (12 g, 19.4 mmol) and tributyltin hydride (6.25 ml, 23.3 mmol) were mixed in a pressure tube, sealed and heated to 80 °C for 2 h with vigorous stirring. The reaction mix was cooled to room temperature and dissolved in hot hexane then filtered hot. The product crystallized upon standing in the fridge and was recrystallized from hot hexane to yield (8.4 g, 17.1 mmol) 88 % pure product as a white powder. NMR (75 MHz, CDCl 3 ): δ = 62.23, 31.92, 30.74, 16.86

	Heptadecafluorododecyl bromide (137) 2
	Heptadecafluorododecan-1-ol (136) 2
	1 H NMR (300 MHz, CDCl 3 ): δ = 3.73-3.76 (m, 2H), 2.06-2.21 (m, 2H), 1.67-1.81 (m, 4H)
	1.54 (s, br, 1H).

1 13 C NMR (75 MHz, 13 C 19 F NMR (400 MHz, CDCl 3 ): δ = -83.34 (t, J = 10.16 Hz, 3F), -116.35 (s, br, 2F), -123.70 (s, br, 2F), -123.34 (s, br, 4F), -124.71 (s, br, 2F), 125.47 (s, br, 2F), 128.25 (s, br, 2F).

  H NMR (300 MHz, CDCl 3 ): δ = 3.47 (t, J = 6.45 Hz, 2H), 2.07-2.19 (m, 2H), 1.95-2.04 (m, 2H), 1.78-1.87 (m, 2H). F NMR (400 MHz, CDCl 3 ): δ = -81.74 (t, J = 8.96, 3F), -115.16-115.38 (m. 2F), 122.67 (s, br, 2F), 122.88 (s, br, 4F), 123.67 (s, br, 2F), 124.44 (s, br, 2F), 127.07 (s, br, 2F).

	Fluorinated gemini (138) 2

1 13 C NMR (75 MHz, CDCl 3 ): δ = 32.40, 31.91, 30.12, 19.12.

19

  H NMR (300 MHz, CD 3 Cl / CD 3 OD): δ = 4.30 (s, 4H), 3.75 (s, 12H), 3.56-3.64 (m, 4H) 3.21-3.38 (m, 4H), 1.87-1.97 (m, 4H), 1.69-1.89 (m, 4H).Kryptopyrrole (1.00 g, 8.12 mmol), perfluorobenzaldehyde (0.80 g, 4.06 mmol) and few drops of TFA were dissolved in DCM (100 mL). The reaction was monitored by TLC and after consumption of the aldehyde, chloranil (1 g, 4.06 mmol) was added. The reaction mixture was stirred for 15 min. Diisopropylethylamine (4.83 mL, 28.4 mmol) was added and the reaction was further stirred for a further 5 min. Boron trifluoride etherate (6.34 g, 44.6 mmol) was added and the reaction mixture was stirred for 1 h. Water was added and the phases were separated. The organic phase was washed with water (3x) and reextracted (1x). The organic phase was dried over MgSO 4 , filtered and evaporated. The crude product was purified by column chromatography using PTE/DCM (70/30, v/v) to yield 0.73 g (1.6 mmol, 38 %) of the product as a golden brown powder. CDCl 3 ): δ = 156.27, 136.70, 134.07, 130.48, 17.24, 14.70, 12.89, 11.01.

	F 5 -ethyl-BODIPY (148) 4
	N + Br -	N +	Br -
	C 8 F 17	C 8 F 17
	1 H NMR (300 MHz, CD 3 OD): δ = 4.10 (s, 4H), 3.56-3.62 (m, 4H), 3.32-3.34, (m, 12H), 2.29-
	2.47 (m, 4H), 1.96-2.07 (m, 4H), 1.71-1.82 (m, 4H).
	Spectra conform with literature

1 1 H NMR (300 MHz, CDCl 3 ): δ = 2.54 (s, 6H); 2.33 (q, 4H, J = 7.58 Hz), 1.51 (s, 6H), 1.02 (t, 6H, J = 7.58 Hz).

13 C NMR (75 MHz,

  NMR (300 MHz, CDCl 3 ): δ = 8.23 (d, J = 17.04 Hz, 1H), 7.72 (d, J = 17.02 Hz, 1H), 6.17 (s, 2H), 3.94 (s, 6H), 3.88 (s, 3H), 3.27 -3.23 (m, 2H), 2.66 (q, J = 7.45 Hz, 2H), 2.49 -2.39 (m, 2H), 2.35 (q, J = 7.51 Hz, 2H), 1.58 (s, 3H), 1.55 (s, 3H), 1.51 (s, 3H), 1.22 (t, J = 7.61 Hz, 3H), 1.02 (t, J = 7.53 Hz, 3H). NMR (75 MHz, CDCl 3 ): δ = 162.01, 160.75, 155.50, 154.24, 148.84, 148.72, 146.38, 146.23, 145.68, 143.18, 143.03, 137.20, 134.75, 134.47, 133.44, 131.78, 130.30, 130.08, 120.58, 119.32, 117.00, 108.83, 90.94, 56.08, 55.54, 32.28, 32.07, 29.86, 25.60, 18.79, 17.29, 14.73, 12.90, 10.70, 10.55.19 F NMR (400 MHz, CDCl 3 ): δ = -81.69 (t, J = 9.92 Hz, 3F), -114.87 --115.04 (m, 2F), -

1 H 13 C 122.59 (s, 2F), -122.83 (s, 4F), -123.63 (s, 2F), -124.36 (s, 2F), -127.03 (s, 2F), -133.26 --133.35 (m, 2F), -139.24 --139.34 (m, 2F), -144.25 --144.51 (m, 2F).

H NMR (300 MHz, CDCl 3 ): δ = 7.79 (s, 1H), 7.49-7.33 (m, 5H), 7.05 (s, 1H), 5.20 (s,

2H),

Acknowledgements

First of all I would like to express my sincere gratitude to my supervisor Dr. Nathan D.

McClenaghan for giving me the opportunity to carry out this doctoral research in his group and the advice and inspiration concerning the research project. Beside the scientific part I

Scheme 16: Synthesis route for the amphiphilic ratiometric Ca 2+ -sensor (97).

Having protected the aldehyde, the benzyl group could be removed using AlCl 3 . The free phenol (90) was obtained in 69 % yield and could be reacted with 4-(benzyloxy)-1-(2bromoethoxy)-2-nitrobenzene under basic conditions to yield a classical BAPTA precursor (91) already bearing the dinitrophenoxyethane fragment. Selective reduction of the nitro

Solvents and chemical products

Commercially-available products were purchased from the Sigma Aldrich, Acros Chemicals, Lancaster and Fluka and were used as received. Solvents of technical grade were distilled and dried prior to utilization. Toluene was distilled over sodium, acetonitrile and dichloromethane were distilled over calcium hydride and dimethylformamide (DMF) was stored over molecular sieves. Deuterated solvents for NMR analysis were bought from Sigma-Aldrich and Euriso-top. Deionized water was obtained by purification over an ion exchange column and a membrane filter of 0.45 µm (MSI, Micron separation, Inc.). Solvents for spectroscopy without the addition of stabilizing agents or other absorbing material were employed as bought.

Thin layer chromatography and silica columns

Thin layer chromatography was performed on silica gel 60 F254 sheets on aluminium produced by Merck. Spots on the TLC plate were observed under UV light (254 nm / 365 nm) when appropriate for non-active compounds an appropriate staining agent were employed.

Most frequently used staining agents comprised an ethanolic solution of fluorescein, dinitrophenylhydrazine staining agent for aldehyes, ninhydrin solution for amines, cerium molybdate staining agent for alcohols and as a general stain and phosphomolybdic acid stain as a general staining agent. Column chromatography for the separation of organic compounds was performed using silica gel from Merck with a particle size of 40 -63 µm (230 -400 mesh). Organic compounds for separation were dissolved in a minimum amount of chromatography solvent and deposited on top of the column and subsequently eluted.

Nuclear magnetic resonance spectroscopy (NMR)

1 H and 13 C-NMR experiments were performed at 295 K on a Bruker Avance 300 ( 1 H: 300 MHz, 13 C: 75 MHz), Avance II 400 ( 1 H: 400 MHz, 13 C: 100 MHz), and an Avance III 600 ( 1 H: 600 MHz, 13 C: 150 MHz) spectrometer. Chemical shifts are reported in ppm (δ) and are referenced to the NMR solvent (CDCl 3 , D 2 O, MeOD, CD 3 CN, DMSO) residual peak. Abbreviations used are s = singlet, d = doublet, t = triplet, q = quartet, dd = doublet of doublets, dt = doublet of triplets and m = multiplet. The coupling constants (J) are reported in Hertz (Hz).

Mass spectrometry

Mass spectra are measured by the "Centre d'Etude Structurale et d'Analyse des Molecules Organique" (CESAMO) at the university of Bordeaux. The measurements were carried out on a QStar Elite mass spectrometer (Applied Biosystems). The instrument is equiped with an electrospray ionisation (ESI) source and the spectra were recorded in either positive or negative ionisation mode. The electrospray needle was maintained at 5000 V and operated at room temperature. Samples were introduced by injection through a 20 µL sample lip into a 4500 µL/min flow of methanol from the LC pump. Alternatively field desorption (FD) was used as ionization mode on a time of flight (TOF) mass spectrometer (Accu TOF GCv). The FD-emitter voltage was 10 kV and 1 -2 µL of sample solution was deposited on a 13 µm emitter wire and inserted into the machine.

Electronic absorption (UV-Vis) and fluorescence spectroscopy

Electronic absorption and emission spectra have been measured on a Varian UV-Vis-NIR spectrophotometer Cary 5000 or Cary 100. Heterogeneous samples were recorded on a Cary 100 equiped with an integrating sphere (labsphere DRA-CA-301). The wavelengths observed ranged from 200 -800 nm. Sample solutions were measured in matched quartz cells with a pathlength of 10 mm. Before each measurement a baseline of pure solvent was been taken and was subtracted from the measured spectra. The fluorescence emission spectra were measured on a HORIBA Jobin-Yvon Fluorolog -3 equiped with a xenon lamp (450 W), with Hamamatsu R2658P and R928P photomultiplier (PMT) detection. Quartz cells of 10 mm length were employed for study of optically dilute samples with fluorescence emission being measured at a right angle with respect to the excitation beam. Time-correlated single photon counting was performed with a monochromatic pulsed light source (nanoLED 370 nm and 456 nm; 1.2 ns FWHM) on the Fluorolog-3. After irradiating a solution of ferrioxalate (3 ml) for a certain time, the solution of phenanthroline (0.5 ml) is added, which forms a tris-phenanthroline complex only with the Fe 2+ ions (ɛ = 11100 cm -1 M -1 at 510 nm) The absorption is compared to a non-irradiated reference solution which is kept in the dark and the difference between the two is noted and gives ∆A. The molar amount of Fe 2+ is given by equation 28.

Photochemical quantum yield.

moles Fe 2+ = ×௱ ×ɛ ఱభబ equation 28

Where V is the total volume (3.5 ml) l is the optical path of cell and ɛ 510 is the extinction coefficient of the Fe(phen) 2+ complex. Thus, the photons absorbed by the solution per time unit (Nhν/t) is:

Where ɸ λ is the quantum yield for the formation of Fe 2+ , t is the irradiation time F is the The x-intercept in this graph gives the logarithm of the K d as shown in figure 119.

Preparation of vesicles

Vesicles were prepared using a popular thin film hydration procedure. A solution of amphiphile and membrane fluorophores or Ca 2+ -probes in a mixture of CHCl 3 and MeOH (3:1, v/v) was prepared and the solvent was slowly evaporated on a rotary evaporator by increasing the vacuum stepwise until a thin film was obtained. After drying the thin film an additional 30 min, it was hydrated using an appropriate buffer (100 mM KCl, 30 mM MOPS, pH 7.2). The obtained polydisperse vesicle solution was passed through a membrane of 800 -200 µm pore size depending on the desired vesicle size.

Preparation of multicompartmentalized polymersomes

A formulation of liposomes in pseudo-intracellular buffer loaded with 130 mg/ml sucrose which equals an osmotic pressure of 380 mOsm is emulsified in toluene using poly(butadiene) 46 -b-poly(ethylene oxide) 30 (PB-b-PEO) (3 mg/ml). The solution was gently shaken to produce droplets of a water-in-oil emulsion. The emulsion was then set on top of an aqueous pseudo intracellular buffer solution containing 380 mOsm of glucose. In a last step, the biphasic system is centrifuged, yielding giant polymersomes with the prepared vesicle solution in the aqueous interior.

Optical and epifluorescence microscopy

Samples were sealed between a glass slide and co Glan-Thompson polarizers. Laser powers were adjusted as described in the result section.

Spectroscopy was performed with an Andor system, collecting after the pinhole were intensity corrected for instrumental response, measuring suitable reference solutions that cover the whole spectral range. A schematic representation of the setup is given in figure 120. Thompson polarizers. Laser powers were adjusted as described in the result section.

Spectroscopy was performed with an Andor system, collecting after the pinhole. The spectra were intensity corrected for instrumental response, measuring suitable reference solutions that cover the whole spectral range. A schematic representation of the setup is given in figure 120.

microscopy setup used in the surface patterning experiments.

Confocal microscopy 2

For confocal fluorescence observations, giant polymersomes were deposited by capillarity in hydrophobic micro-channel microscopy slides, µ-Slide VI

Dynamic light scattering

The size of the vesicle formulations had been measured using a Malvern CGS-3 dynamic light scattering machine equipped with an ALV correlater. ALV5000EPP and ALV60x0 Software were used to determine the vesicle size with a laser beam at 632 nm measured at an angle of 90° at room temperature.

Synthesis

F 5 -BODIPY (59) In a round bottom flask, perfluorobenzaldehyde (0.5 g, 2.5 mmol) was dissolved in freshly distilled dichloromethane (35 mL). 2,4-dimethylpyrrole (0.8 mL, 7.8 mmol) and a drop of TFA were added and the mixture was stirred for 4 h. p-Chloranil (0.62 g, 2.5 mmol) in solution in CH 2 Cl 2 (20 mL) was added dropwise and the mixture was stirred for 1 h at room temperature.

Then triethylamine (5.0 mL) and borontrifluoride etherate (5.0 mL) were added and the mixture was stirred at room temperature two additional hours. Water was added and the product was extracted with CH 2 Cl 2 , the organic phase was dried over MgSO 4 and the solvent was evaporated. The crude product was purified by chromatography PTE / EtAc (90/10, v/v)