Development of Photoactivatable Compounds

MASARYK UNIVERSITY

FACULTY OF SCIENCE

DEPARTMENT OF CHEMISTRY

Development of

Photoactivatable Compounds

Ph.D. Thesis

Lenka Filipová

SUPERVISOR: prof. RNDr. Petr Klán, Ph.D. BRNO 2018

Bibliographic Entry

Author: Mgr. Lenka Filipová

Masaryk University, Faculty of Science

Department of Chemistry

Title of Dissertation: Development of Photoactivatable Compounds

Degree Programne: Chemistry

Field of Study: Organic chemistry

Supervisor: prof. RNDr. Petr Klán, Ph.D.

Masaryk University, Faculty of Science

Department of Chemistry

Academic Year: 2018/2019

Number of Pages: 129 pages + 109 pages of appendices

Keywords: reverse micelles, azobenzene, strained alkynes, cyclopropenone,

Cu-free click reaction, photoCORMs, heptamethine cyanine dyes, Zincke reaction

Bibliografický záznam

Autor: Mgr. Lenka Filipová

Masarykova univerzita, Přírodovědecká fakulta

Ústav chemie

Název práce: Vývoj fotoaktivovatelných sloučenin

Studijní program: Chemie

Studijní obor: Organická chemie

Školitel: prof. RNDr. Petr Klán, Ph.D.

Akademický rok: 2018/2019

Počet stran: 129 stran + 109 stran příloh

Klíčová slova: reverzní micely, azobenzen, napnuté alkyny, cyclopropenon,

Cu-free Click reakce, photoCORMy, barviva heptamethine

cyaninová, Zincke reakce

Abstract

My dissertation thesis was focused on the synthesis of new photoactivatable compounds and studying their photochemical properties and behavior. The results summarized in this thesis have been published or will be submitted for the publication in the near future.

The introductory part of the dissertation thesis provides previously published information related to the studied projects, including properties and behavior of reverse micelles, photoisomerization of azobenzene derivatives, click chemistry of strained alkynes, followed by application of cyclopropenones for photochemical generation of strained alkynes.

Finally, the role of carbon monoxide as a signaling molecule and its potential biological effects are described. Previously reported carbon monoxide releasing molecules are also shown. In the last section of this chapter, preparation and applications of heptamethine cyanine dyes are summarized

In the next chapter, Results and Disccusion, results of three research projects are summarized. The first of them is focused on the development of new photoactivatable amphiphiles and their utilization in preparation of reverse micelles. Properties of photoresponsive amphiphiles and the formation reverse micelles were studied by NMR spectroscopy at both room and sub-zero temperatures. The second project was focused on photochemically initiated Cu-free click reaction, which is widely used in the conjugation of biomolecules in the absence of metals used as catalysts. A new silacyclopropenone was developed and used in a subsequent, photoinduced Cu-free click reaction with azides and tetrazines. The third project was focused on the synthesis of new heptamethine cyanine dyes.

During development of new photoactivatable compounds absorbing at higher wavelengths,

Zincke reaction was utilized in the synthesis of various cyanine dyes substituted on the heptamethine chain. This method allowed the installation of different functional groups and synthesis of heptamethine cyanine dyes inaccessible by known methods.

In the last part of thesis, the publications and experimental data are appended.

Abstrakt

Má doktorská práce byla zaměřena na syntézu nových fotoaktivovatelných sloučenin a studium jejich fotochemických vlastností. Získané výsledky popsané v této práci již byly publikovány nebo jsou sepsány a připraveny k publikaci.

V úvodní kapitole jsou shrnuty dříve publikované relevantní informace, týkající se studovaných projektů, zahrnujících vlastnosti a chování reverzních micel, photoisomerizace azobenzenových derivatů, click reakce a využití napjatých alkynů a cyclopropenonů pro fotochemickou tvorbu napjatých alkynů. Dále je popsána tvorba oxidu uhelnatého jako signální molekuly v biologických systémech a jeho biologické účinky. V této části se diskutují známé molekuly uvolňující oxid uhelnatý. V poslední části této kapitoly jsou shrnuty poznatky o cyaninových barvivech, způsoby jejich přípravy a jejich využití.

V následující kapitole Výsledky a diskuze jsou ukázány a shrnuty výsledky tří výzkumných projektů. První z nich se věnuje vývoji nových fotoaktivovatelných amfifilů a jejich využití při tvorbě reverzních micel. Vlastnosti samotného amfifilu a z něj vytvořených reverzních micel byly studovovány především pomocí NMR spektroskopie za laboratorní a nízké teploty. Druhý projekt se věnuje click reakci probíhající za nepřítomnosti mědi, reakci, která je využívána pro spojování biomolekul v nepřítomnosti kovů používaných jako katalyzátory. Nový silacyclopropenon byl vyvinut a použit ve světlem iniciované click reakci s azidy a tetraziny jako reagenty. Třetí projekt se zabývá syntézou nových různě substituovaných heptamethinových cyaninových barviv pomocí Zinckeho reakce. Tato metoda umožňuje instalaci mnoha funkčních skupin a syntézu cyaninových barviv, která dosud nebyla dostupná známými metodami.

V poslední části práce jsou přiloženy publikace a experimentální data.

Acknowledgement

I would like to thank prof. Petr Klán for his supervision and opportunity to work in his group. I am grateful for fruitful discussions, support, motivation and especially for his trust in my abilities.

Much thanks goes to my partner Peter. I cannot express by words how grateful I am to have you, but I hope you know it. You helped me during my entire doctoral studies, even despite the large distance between us. It was not always easy, but you stayed with me every time and endured my bad mood or crying. You are my teacher, my best friend and my husband soon. I am so grateful that we can work together and share our laboratory experience, as well as our life together. We are perfect collaborators, not only in the laboratory but also in our lives, and I hope that our collaboration will last forever. I truly and deeply love you.

I thank to my current colleagues, namely Estelita, Marina, Qiuyun, Andreas, Sadegh,

Mišo, Tomáš and Marek for their fruitful discussions not only about chemistry and for creating an inspiring atmosphere and fun during the work. I never experienced so nice, kind and funny group of people who like each other and work together. I am grateful that all of you are my colleagues and especially my friends.

I also thank my colleagues and friends which I met during my study, Tombo and Lulu.

Thanks to Luboš for technical support and providing us with everything which we ever needed in laboratory.

Last but not at least I thank to my family for their support during my study, especially when they could not comprehend how I could study chemistry. Without you I would never be the person which I am today.

V neposlední řadě děkuji své rodině za jejich podporu během celého studia, obzvlášť když nikdy nechápali, jak mohu studovat zrovna chemii. Vím, že to se mnou často nebylo jednoduché, ale vy jste tu vždy byli pro mě, snažili se mi všemožně pomáhat a být mi oporou.

Bez vás bych nikdy nebyla tou osobou, kterou jsem dnes.

“Without theory to guide him the

experimenter is as lost as a sailor setting

out without compass or rudder.”

‒ Leonardo da Vinci

“I put my heart and my soul into

my work, and I lost my mind in the

process.”

‒ Vincent van Gogh

Table of Contents

Table of Contents ...... 8

1. Literature Part ...... 11

1.1. Introduction ...... 11

1.2. Amphiphiles and its Aggregates in the Solution...... 11

1.2.1. Reverse Micelles (RMs) ...... 13 1.2.2. Mechanism of Micelle Formation ...... 15 1.2.3. Determination of Critical Micelle Concentration (CMC) ...... 16 1.2.4. Size of Reverse Micellar Aggregates ...... 17 1.2.5. Utilization of Reverse Micelles ...... 17 1.2.6. Reverse Micelles at Subzero Temperatures ...... 17 1.3. Photoswitchable Molecules ...... 20

1.3.1. Azobenzene Photochemistry ...... 20 1.3.2. Preparation of Azobenzene and its Derivatives ...... 21 1.3.3. Azobenzene Derivatives ...... 22 1.3.4. Azobenzene Moiety Incorporated into a Self-Assembly ...... 22 1.4. Click Chemistry ...... 24

1.4.1. Bioorthogonal Click Reaction (Cu-free Click Reaction) ...... 25 1.4.2. Strained Alkynes ...... 26 1.4.3. Cyclopropenones...... 28 1.5. Carbon Monoxide–Releasing Molecules (CORMs) ...... 32

1.5.1. CORMs Based on Solvent-Induced Ligand Exchange ...... 35 1.5.2. Enzyme-Triggered CORMs (ET-CORMs) ...... 35 1.5.3. Photochemically Triggered CORMs (PhotoCORMs) ...... 35 1.5.4. Phototherapeutic Window ...... 37 1.6. Cyanine Dyes ...... 39

1.6.1. Classic Polymethine Cyanine Dyes ...... 39 1.6.2. Trimethine and Pentamethine Cyanine Dyes (Cy3 and Cy5) ...... 40 1.6.3. Heptamethine Cyanine Dyes (Cy7) ...... 42

Table of Contents

1.6.4. Zincke Reaction ...... 42 1.6.5. Photochemistry and Photooxidation of Cyanine Dyes ...... 44 1.6.6. Utilization of Cyanines ...... 45 2. Results and Disccusion ...... 47

2.1. Photoswitching of Azobenzene-Based Reverse Micelles above and at Subzero

Temperatures As Studied by NMR and Molecular Dynamics Simulations ...... 47

2.1.1. Introduction ...... 47 2.1.2. Results and Discussion ...... 47 2.1.3. Synthesis ...... 47 2.1.4. Photoisomerization of Final Azobenzene-Containing Amphiphile ...... 48 2.1.5. Reverse Micelles Formation ...... 50 2.1.6. General Procedure for Preparation of Reverse Micelles ...... 51 2.1.7. Determination of Critical Micelle Concentration ...... 51 2.1.8. Photoisomerization of Reverse Micelles at 303 K ...... 54 2.1.9. Molecular Dynamic Simulations (work of E. Muchová and P. Slavíček) ...... 57 2.1.10. Reverse Micelles at Subzero Temperatures ...... 58 2.1.11. Photoisomerization of Reverse Micelles at Subzero Temperatures ...... 59 2.1.12. Conclusions ...... 61 2.1.13. Author´s Contribution ...... 61 2.2. Photochemical Formation of Dibenzosilacyclohept-4-yne for Cu-Free Click

Chemistry with Azides and 1,2,4,5-Tetrazines ...... 62

2.2.1. Introduction ...... 62 2.2.2. Synthesis (work of J. Galeta) ...... 63 2.2.3. Photochemistry of Silacyclopropenone...... 63 2.2.4. Irradiation of Silacyclopropenone in the Presence of Benzyl Azide ...... 64 2.2.5. Cu-free Click Reaction of Silacyclopropenone with Tetrazines ...... 67 2.2.6. Conclusion...... 69 2.2.7. Author´s Contribution ...... 69 2.3. Development of New PhotoCORMs based on Heptamethine Cyanine Dyes ...... 70

2.3.1. Introduction ...... 71 2.3.2. Synthesis ...... 73 2.3.3. Zincke Reaction ...... 75

Table of Contents

2.3.4. Photochemistry of Target Carboxylic Acids ...... 77 2.3.5. CO Determination ...... 78 2.4. Cyanine Dyes Substituted at the Heptamethine Chain Accessed by Ring-Opening of

Pyridnium Salts ...... 80

2.4.1. Introduction ...... 80 2.4.2. Optimization of the Reaction Conditions ...... 81 2.4.3. Functionalization of the Pyridinium Salts ...... 83 2.4.4. Monosubstituted Cyanine Dyes ...... 84 2.4.5. Disubstituted Cyanine Dyes ...... 87 2.4.6. Up-scale and Modification of Heterocycles ...... 88 2.4.7. Photochemical Characterization of the Prepared Cy7 Dyes ...... 88 2.4.8 Conclusion...... 92 2.4.9. Author´s Contribution ...... 92 3. Experimental Part ...... 81

3.1. Materials and Methods ...... 81

3.2. Synthesis ...... 94

4. Conclusion ...... 112

5. References ...... 113

6. List of Abbreviations ...... 125

7. Curriculum Vitae ...... 127

8. List of Appendices ...... 129

Appendix 1A: Langmuir 2017, 33, 2306-2317 ...... 130

Appendix 1B: Langmuir 2017, 33, 2306-2317 – Supporting Information ...... 143

Appendix 2A: Org. Lett. 2016, 18, 4892-4895 ...... 168

Appendix 2B: Org. Lett. 2016, 18, 4892-4895 – Supporting Information ...... 173

Appendix C: NMR Spectra of Compounds from Chapter 2.3. and 2.4...... 192

Literature Part – Amphiphiles

1. Literature Part

1.1. Introduction

Photochemistry is a part of chemistry where the light is used as a source of energy.

Our major interest is focused on visible light, which has the absorption in the range of 400-700 nm (Figure 1). In biological applications, ultraviolet light of higher energy may damage biological tissues and cells. The use of light provides an opportunity for spatial and temporal control of photochemical reactions. In the recent years,1–3 the research has often been focused on the development of new photoactivatable compounds, which are potentially applicable in biological environment for bioimaging and targeted deliveries. Therefore, the development of new chromophores absorbing the light in the visible and near-infrared region, where the biological tissues and cells do not absorb, is essential.

Figure 1. Electromagnetic spectrum with highlighted visible light area.

1.2. Amphiphiles and its Aggregates in the Solution

Amphiphilic molecules, also called amphiphiles or surfactants, are molecules, which contain polar hydrophilic and nonpolar hydrophobic parts (also called hydrophilic head and hydrophobic tail) (Figure 2). The most common and commercially available amphiphilic molecules are cationic surfactants CTAB 1 and CTAC (cetyltrimethylammonium bromide and chloride, respectively)4 and anionic AOT 2 (Aeroasol OT = sodium bis(2-ethylhexyl)sulfosuccinate)5.

Literature Part - Amphiphiles

Figure 2. Chemical structure of cationic CTAB 1 and anionic AOT 2.

Amphiphilic molecules exhibit a unique behavior in different solvents and can self-assemble into larger, organized structures (Figure 3). In polar solvents such as water, hydrophobic tails are oriented into the assembly to minimize the entropy penalty of the water confinement, thus micelles, vesicles, bilayers or micellar rods can be formed. The molecular structure of a surfactant controls the shape and size of the resulting aggregates and the shape can be predicted thanks to the calculated packing parameter.6 The packing parameter P is defined as

푷 = 풗풕⁄풂풉풍풕 where 푣푡 and 푙푡 are the volume and the length of the surfactant tail and 푎ℎ is the optimal head group area.7

The volume of the chain 푣푡 can be calculated from the number of carbon atoms n in a saturated hydrocarbon chain according to the expression8:

ퟑ 풗풕 = ퟐퟕ. ퟒ + ퟐퟔ. ퟗ풏 (Å )

Length of the surfactant tail 푙푡 is limited by the 푙푚푎푥, which is the maximum possible extension of a hydrocarbon chain. This distance is obtained from the distance 2.53 Å between alternate carbon atoms of a fully extended chain with the addition of the van der Waals radius of the terminal methyl group equal to 2.1 Å. The length of the surfactant tail 푙푡 is defined as:

ퟑ 풍풕 ≤ 풍풎풂풙 = ퟏ. ퟓ + ퟏ. ퟐퟔퟓ풏 (Å )

The optimal head group area 푎ℎ is determined by the head interactions and can be estimated from the chemical potential of a surfactant molecule in the micelle.9 The head contribution to the chemical potential is the sum of van der Waals attraction and electrostatic repulsion.

Literature Part – Amphiphiles

If the packing parameter P is smaller than 0.33, then the surfactant has cone critical packing shape, resulting in the formation of spherical micelles (Figure 3). With P in the range of 1/3 to 1, the critical packing shape of the truncated cone causes the formation of cylindrical micelles, rods and flexible bilayers and vesicles. If the is P ~ 1, planar bilayers are formed in the solution. The critical packing shape of inverted truncated cone with P > 1 permits formation of inverted (reverse) micelles. The reverse micelles are formed in non-polar solvents

(and oils), where the hydrophilic heads and hydrophobic tails points inwards and outwards of the aggregates, respectively.

Surfactant molecules are also parts of microemulsion. The microemulsion is a system of surfactant, non-polar solvent (oil) and water, which is a single optically isotropic and thermodynamically stable liquid solution.10 Conic surfactants with a P < 1 assembled into an oil-in-water microemulsion (o/w), where oil phase is encapsulated inside the aggregates in water. For surfactants with a P > 1, the water-in-oil microemulsion (w/o) is formed and water is enclosed inside the assembly. Surfactants with P ~ 1 form the lamellar phases, where the bilayers of surfactants are created.

Figure 3. Predicted shapes of aggregates based on the critical packing parameter P.

1.2.1. Reverse Micelles (RMs)

Surfactants dissolved in organic non-polar solvents form spherical reverse micelles.

They can be formed in the presence as well as in the absence of water.11 However, if the medium is completely free of water, the aggregates are very small and polydisperse.

The presence of water is necessary for formation of larger reverse aggregates. Water is

Literature Part - Amphiphiles solubilized in the polar core and the water pool inside the assembly is formed.12,13

The aggregates display a multilayer structure consisting of interfacial and core water (bulk water) as shown in Figure 4.

Figure 4. Schematic illustration of a multilayer structure of the reverse micelles.

The amount of water inside the reverse micelles is characterized by the water-to-surfactant molar ratio x and the water loading w, which are defined as14:

풄(풘풂풕풆풓) 풙 = 풄(풔풖풓풇풂풄풕풂풏풕)

풄(풘풂풕풆풓 풊풏 풕풉풆 풄풐풓풆) 풘 = 풄(풔풖풓풇풂풄풕풂풏풕 풎풐풍풆풄풖풍풆풔 풐풇 풕풉풆 풂풔풔풆풎풃풍풚)

Thus, if all the water molecules are incorporated into the structure of reverse micelles, the x and w values are equal.

The size of reverse micelles can be controlled by varying the water-to-surfactant molar ratio x. In one of the earliest studies,15 the reverse micelles were divided into 3 groups according to the water loading: small micelles (w < 7), medium size micelles (10 < w < 30) and large micelles (w > 30). In other study,16,17 the incorporated water in reverse micelles was divided into three states according to the water loading w: for w > 10, where water is considered as an interfacial layer and it is directly bound to the surfactant headgroups of an amphiphile. In the second group where water loading is in the range 10 < w < 30, water molecules migrate towards the center of reverse micelles with properties that resemble those of bulk water. For w > 30, the micellar water has the same static and dynamic properties as bulk water or “free water”.

Literature Part – Amphiphiles

Several studies were performed with reverse micelles with various water loadings and radii. The surfactant AOT forms well characterized spherical reverse micelles in isooctane over a large range of water content from very small micelles with w = 2 (1.7 nm diameter),18 through larger micelles with w = 25, 37 or 46 with radii 4.5, 8.5 and 10 nm, respectively,19 up to large micelles with ~ 60 water molecules per AOT.20 The smallest reverse micelles have radii of around 1 nm (50-100 water molecules per micelle), whereas the largest micelles have radii of up to 14 nm (~400 000 waters per micelle).19

On the contrary, CTAB reverse micelles are much less studied compared to AOT, and the amount of water assembled inside the reverse micelles is variable depending on the environment. For instance, in CTAB/isooctane/n-hexanol/water reverse micelles, the water loading w ranged from 5 to 40.21 However, in CTAB/chloroform-d/water system, the maximum water loading w was found to be 3.4.14

1.2.2. Mechanism of Micelle Formation

Micellization is a process of the formation of larger particles from monomer units.

Various models of micellization have been proposed in the literature. Two classical models are widely used for the characterization of the aggregates – open22 and closed23 association models. The open association model describes a continuous growth of aggregates and implies a step-by-step micellar growth. Therefore, an ensemble of species with different degrees of aggregation is present in the solution. The closed association model, also called mass action model, presumes a dynamic equilibrium between the monomers and the molecules associated in the aggregates. The closed model involves a well-defined critical micelle concentration

(CMC), at which dramatic changes of physical properties occur. On the other hand, absence of a well-defined CMC is characteristic for the open model.

In the 1975, Eicke proposed the model of amphiphile aggregation in non-polar solvents.24 Eicke’s association model has been recently confirmed in a study of micellization in a CTAB/water/chloroform-d system.14 This model describes organization of monomer units in the solution. At very low concentration, only the monomer units are present in the solution.

With increasing concentration, the monomer units are connected together and form linear premicellar aggregates. When the concentration reaches the point called critical micelle concentration (CMC), the premicellar aggregates form the reverse micelles (Figure 5).

Literature Part - Amphiphiles

Figure 5. Reverse micelles formation in the CTAB/water/chloroform-d system according to

Eicke‘s association model. Reproduced with permission from Langmuir 2012, 28, 15185-15192.

1.2.3. Determination of Critical Micelle Concentration (CMC)

The critical micelle concentration is an important characteristic in the reverse micelle formation studies. The determination of the CMC is based on measurement of a drastic change of physical properties. Various methods have been employed for this purpose, such as

1H NMR,25 isothermal titration calorimetry,26 conductometry,27 fluorescence measurement,28 etc.

1H NMR spectroscopy provides information about the character of water in the cationic reverse micelles present in the non-polar solvents such as chloroform and n-hexane.29

Typical chemical shift of water dissolved in CDCl3 has been reported to be 1.56 ppm.30

Downfield chemical shift of water protons, up to 4.00 ppm, reflects the formation of hydrogen bonds in a growing water pool inside the assemblies.31 Moreover, the chemical shift of water proton at 4.79 ppm corresponds to the bulk water.30 It was found in freshly prepared samples prior to equilibration of the system.

According to the close association model, 1H NMR spectroscopy can be used for determination of the CMC. It is determined based on the dependence of the chemical shift

25 of water, 훿표푏푠(퐻2푂), on the total surfactant concentration. A plot of 훿표푏푠 as a function of 1⁄푐 should consist of two straight lines, one below and one above the CMC. The intersection point of the two lines corresponds to 1/CMC.

Literature Part – Amphiphiles

1.2.4. Size of Reverse Micellar Aggregates

Various methods are useful in the determination of size and shape of assemblies, e.g. dynamic light scattering (DLS),32 small angle X-ray scattering (SAXS),33,34 small angle neutron scattering (SANS),35 transmission electron microscopy36 and diffusion 1H NMR.37

Nuclear magnetic resonance (NMR) provides a tool to establish the self-diffusion coefficients using a pulsed gradient spin echo (PGSE) sequence in 1H NMR diffusion experiments.37,38 The hydrodynamic radius RH of spherical aggregates can be calculated from the experimentally determined diffusion coefficients using Stokes-Einstein equation:

풌푩푻 푹 = 푯 ퟔ흅휼푫 where 푘퐵 is the Boltzmann constant, η is the solvent viscosity and D is the diffusion coefficient of aggregates.

1.2.5. Utilization of Reverse Micelles

Reverse micelles are used in many applications and they are the subject of interest in many fields of science. The reverse micelles are used for preparation of semiconductors,39 synthesis of inorganic nanomaterials40 or media for nanoscale polymerization reaction, which provides the polymers with a controlled conjugation length.41

One of the most interesting applications of these systems comes from biology and biotechnology. Reverse micelles can solubilize proteins and other hydrophilic molecules in organic solvents due to the presence of aqueous microenvironment in the assembly.42 Some proteins, such as α-chymotrypsin, were found to be highly stable in a reverse micellar system.43,44 The reverse micellar systems are also used as a model for biological systems,45 extraction and purification of biomolecules,46,47 drug delivery,48 and also as enzymatic reaction media.49

1.2.6. Reverse Micelles at Subzero Temperatures

The behavior of water inside reverse micelles has been the subject of many studies in the recent years.50 Three distinct processes have been identified at low temperatures – the presence of supercooled water, water shedding and formation of ice inside the reverse micelles.

Literature Part - Amphiphiles

Supercooling is a state where liquids do not solidify even below their normal freezing point. A supercooled state of water can be generated in thin films,51 capillary samples,52 droplet samples,16 and emulsion,53 under conditions that minimize homogenous nucleation.50

In the reverse micelle system AOT/pentane/solution of NaCl,50 the formation of a supercooled water in small volumes was observed with the temperature highly decreasing below 0 °C.

With the decreasing temperature, hydrogen bonding interactions among the water molecules are optimized, leading to the four-coordinate geometry of the ice of an open network character.54,55 It was found, that water inside reverse micelles can be supercooled to at least -39.5 °C at 1 atm. The phenomenon was also observed in small reverse micelles with low w at lower temperature.56

The formation of reverse micelles and encapsulation of water into its structure is entropically driven.57,58 However, the entropically favorable encapsulation does not always contribute enough to the Gibbs free energy to keep reverse micelles undisturbed. At low temperature, the system of reverse micelles is destabilized and the instability causes an effect known as water shedding. This involves expulsion of the water content from reverse micelles until a new equilibrium is established.50,59 The water shedding was observed for anionic AOT reverse micelles at -20 and -30 °C,50 and also for cationic CTAB reverse micelles at -20 °C.60 It was also shown, that water loading is dependent on the amount of water in the reverse micelles. AOT in n-pentane with w = 5 formed a stable structure at - 35 °C, whereas the water was expelled from the aggregates with w above 10.59 This effect of water shedding can be suppressed by fast cooling at sufficiently low temperature.60,61

In some studies, formation of ice inside reverse micelles at sub-zero temperatures was observed. The water core of AOT reverse micelles in n-heptane with x = 5 started to freeze at

– 35 °C and complete freezing was observed at – 55 °C.59 In a different study, AOT reverse micelles with w ≤ 3.5 and up to 150 water molecules formed an amorphous form of ice at

– 93 °C (180 K) and the structure of reverse micelles was not affected during fast freezing.62

Inside the small clusters, there is not enough space for crystallization of ice, therefore, the formation of amorphous ice instead of its crystalline form is observed.63 A minimum amount of water for formation of crystalline ice at sub-zero temperature was established to be above

450 water molecules.64 Recently, the metastable cubic ice was detected in the AOT reverse

Literature Part – Amphiphiles

micelles with a radius RW ≤ 2 nm, whereas the reverse micelles smaller than 1 nm formed amorphous ice.65

Literature Part – Photoswitchable Molecules

1.3. Photoswitchable Molecules

A molecular switch is a molecule, which can be reversibly switched between two or more structural states. Many stimuli have been used for switching, such as pH change,66 temperature,67 or electrochemical stimuli.68

Photoswitches are a subcategory of molecular switches, which use light as a source of energy for their activation. Members of this class include azobenzene, diarylethene, stilbenes, spiropyrans and so forth. The photochemical process is generally clean, without introduction of any additives and reagents to trigger the transformation.

1.3.1. Azobenzene Photochemistry

Azobenzene is a molecule, which undergoes the E–Z (trans–cis) isomerization initiated either photochemically or thermally (Scheme 1). Azobenzene predominantly exists as an

E-isomer 3 in the dark and this form is more stable by approximately 10-12 kcal mol–1 then a

Z-isomer 4.69 The Z-isomer is formed upon irradiation with UV light and is less stable due to the steric hindrance between the two phenyl rings (bent structure). It converts spontaneously back to a thermodynamically more stable E-isomer 3 (planar structure). Therefore, the backwards Z→E isomerization occurs both in the dark and upon irradiation.70,71

Scheme 1. Isomerization of azobenzene to give the planar E- and bent Z- isomers.

The absorption spectrum of azobenzene is characterized by two absorption bands which correspond to the 휋 ⟶ 휋∗ and 푛 ⟶ 휋∗ transitions (Figure 6).72 The stronger absorption band at 320 nm corresponds to the symmetry-allowed 휋 ⟶ 휋∗ transition of the E-isomer 3

(black line). In case of the Z-isomer 4, the 휋 ⟶ 휋∗ transition band is slightly hypsochromically shifted to 280 nm (grey dashed line). A much weaker band at 450 nm corresponds to the symmetry-forbidden 푛 ⟶ 휋∗ transition and this absorption band is much more pronounced for the Z-isomer 4.73

Literature Part – Photoswitchable Molecules

Figure 6. UV-Vis absorption spectra of the E- and Z-isomers. Reproduced with permission

from Angew. Chem. Int. Ed. 2015, 54, 11338-11349.

Azobenzene and its derivatives are interesting and important compounds that have been utilized in many fields of chemistry such as dyes and pigments,74 molecular switches,75 molecular machines,76,77 surface modified materials,78 protein probes,79 holographic recording devices,80 nanotubes,81 micelles,82 etc.

1.3.2. Preparation of Azobenzene and its Derivatives

A typical procedure for the synthesis of azobenzene derivatives is an azo-coupling reaction, which is an electrophilic aromatic substitution with diazonium salts (Scheme 2). The majority of nonsymmetrical azobenzene derivatives are prepared via this reaction, starting from initial diazotization of a primary amine 5 at low temperature.83 The diazonium salts 6 are generally stable only at low temperatures, usually below 5 °C. At higher temperatures, the salt decomposes with a concurrent release of N2. On the other hand, tetrafluoroborate salts of diazonium can often be isolated and are stable at room temperature.84 Electrophilic aromatic substitution of electron rich benzene ring (such as phenol or aniline) with the electrophilic nitrogen of diazonium salt then produces the target azobenzene derivatives.

Scheme 2. Electrofilic aromatic substitution of electron rich benzene ring with diazonium

salt to produce azobenzene derivatives.83

Literature Part – Photoswitchable Molecules

The other synthetic methods for azobenzene preparation include Mills reaction

(condensation of aniline 8 with nitrosobenzene derivatives – Scheme 3),85 Wallach rearrangement of azoxybenzenes, reduction of nitroaromatic compounds with reducing agents, or oxidation of primary aromatic amines with oxidizing agents.83

Scheme 3. Azobenzene derivative 9 prepared via Mills reaction.85

1.3.3. Azobenzene Derivatives

The substituents on the azobenzene core exhibit dramatic influence on the absorption, emission and photochemical properties of the azobenzene as well as stability of both E- and

Z- isomers (Figure 7).86 The substitution of azobenzene with an electron-donating group on one phenyl ring and an electron-withdrawing group on the other one drastically decreases stability of the Z-isomers 10 and 11 since the molecules undergoes efficient thermal Z→E isomerization. The short lifetime of Z-isomers is in order of milliseconds up to minutes due to the presence of a push-pull system.87,88

On the contrary, azobenzenes substituted with electron-donating groups on the both benzene rings, such as 4,4´-dialkoxyazobenzene 12, display a very slow reverse isomerization with the lifetime of Z-isomer in the order of hours (Figure 7).89

Figure 7. The lifetime of selected substituted azobenzenes.71

1.3.4. Azobenzene Moiety Incorporated into a Self-Assembly

Azobenzene moiety can be incorporated into an amphiphilic structure as a part of the lipophilic chain to form photoresponsive amphiphiles. These amphiphiles can subsequently self-assemble into photoresponsive aggregates. Many applications have been reported, such

Literature Part – Photoswitchable Molecules as the formation of light-responsive micelles studied by atomic force microscopy90 and scanning electron microscope91 techniques, light switchable vesicles,92 and block copolymer vesicles dissociated and reformed by light.93

The main interest in the case of photoresponsive amphiphiles is to change a property of the assemblies as a result of E-Z photoisomerization of the azobenzene moiety. Many light- induced property changes are related to the disruption of the azobenzene-contaning aggregates, such as a decrease in dynamic surface tension94 or a viscosity change.95 In a similar fashion, photoresponsive micelles96 and vesicles97 can be reversibly disrupted and reformatted by changing the wavelength of irradiation. Azobenzene amphiphiles have also been used in the targeted disruption of giant unimolecular vesicles98 or for thermo- and phototriggered microgels,99 aggregation and disaggregation of DNA conjugates,100 and photoresponsive nanostructures for drug delivery.101

Literature Part – Click Chemistry

1.4. Click Chemistry

In chemical synthesis, the „click chemistry“ involves reactions which have to fulfill a set of strict criteria. This term was introduced by Sharpless in 2001 to describe the reactions, which are fast, highly selective, high yielding, wide in scope and easy to perform.102 The products should be stable and easily isolated without need of column chromatography. Click reactions occur in a one pot arrangement and should include simple reaction conditions (the process should be insensitive to the presence of oxygen and water) and generate minimal amount of harmful byproducts to be subsequently easily removed. The click chemistry found applications in biochemistry,103 molecular biology,104 materials science,105 polymer chemistry106 or pharmaceutical sciences.107

Cycloadditions were found to be ideal reactions for click chemistry due to their inherent selectivity. In 1893, the first synthesis of 1,2,3-triazoles from acyclic alkyne and phenyl azide has been reported, later known as a Huisgen 1,3-dipolar cycloaddition.108

However, the reaction was slow, gave a mixture of regioisomers and required the temperature over 100 °C; thus such conditions did not fulfill the criteria for a click reaction. Later, the Cu- catalyzed Huisgen reaction has been developed and this reaction gave only one regioisomer as a product.109,110

The classic copper-catalyzed alkyne-azide cycloaddition (CuAAC) is the type of

Huisgen 1,3-dipolar cycloaddition based on formation of 1,4-disubstituted 1,2,3-triazoles 15 from terminal alkyne 13 and azide 14 in the presence of copper (Scheme 4).111

Scheme 4. Schematic illustration of copper-catalyzed azide-alkyne click reaction.

The reaction proceeds in various solvents (DMSO, THF, acetone or water-alcohol mixture), it is tolerant to a wide range of pH (4 – 12) and functional groups, and the pure products are usually isolated by simple filtration or extraction. The active catalyst is Cu(I), which can either be generated in situ from Cu(II) salts via reduction with sodium ascorbate111 or used directly.110 The 1,2,3-triazoles are formed during cycloaddition and the proposed

Literature Part – Click Chemistry catalytic cycle is shown in Scheme 5.112 The produced triazoles are virtually chemically inert to reactive conditions, such as oxidation, reduction or hydrolysis.

Scheme 5. The proposed catalytic cycle of alkyne-azide cycloaddition.

1.4.1. Bioorthogonal Click Reaction (Cu-free Click Reaction)

Due to the substantial aforementioned benefits, the click chemistry is routinely applied in biological systems. When the reaction occurs inside living systems without interfering with the native biochemical processes, the reaction is called a bioorthogonal reaction.113

The bioorthogonal reaction has to fulfil a number of requirements, such as selectivity, biological and chemical inertness, the reaction must be fast to prevent the damage and side reactions in the biological environment. Reaction also has to be a biocompatible, which means non-toxic and has to be carried out under biologically relevant conditions (aqueous solution, pH, temperature).

The classic copper-catalyzed alkyne-azide cycloaddition is not suitable for use in living cells due to the toxicity of Cu(I) ions. 114 In biological system, copper can catalyze the formation of highly reactive hydroxyl radicals, which are responsible to oxidative damage.

On the other hand, the azide is one of the most popular bioorthogonal functional group. To circumvent the need toxic Cu(I) ions, strained alkynes were developed instead of acyclic alkynes for the “copper-free click reactions”.

Literature Part – Click Chemistry

1.4.2. Strained Alkynes

Although the linear alkynes react with azides only at elevated temperatures or under

Cu-catalysis, cycloalkynes react readily with the same substrate at room temperature.115

Cyclooctyne is the smallest cycloalkyne, which was isolated as a stable compound.116

The reactivity of cyclooctyne is caused by its highly strained structure. The bond angles of the sp-hybridized carbons in cyclooctyne are ~ 160°, and approximately 18 kcal mol-1 of ring strain is released in the cycloaddition reaction with azides. Smaller strained alkynes, such as cycloheptyne, cyclohexyne and cyclopentyne, are not stable and have never been isolated and exist only as transient intermediates or as ligands coordinated to a metal center.116,117

It was shown, that the substituted cyclooctyne 16 undergoes spontaneous cycloaddition with various azides at room temperature. It has been applied for modification of biomolecules and living cells without any physiological harm (Scheme 6).118

Scheme 6. Bioorthogonal reaction of substituted cyclooctyne and benzyl azide.

In order to increase the rate constant of the reaction, cyclooctyne was modified with various substituents. Addition of the fluorine atom into the cyclooctyne structure in 19 increased the reaction rate constant by 3-fold (compared to that of 18, Figure 8).119 The DIFO compound 20 with two fluorine atoms reacts with second-order rate constant, which is ~60 times higher than that of the parent cyclooctyne.120 The reactivity of cyclooctyne can also be increased by increasing the ring strain by addition of a phenyl or cyclopropyl moiety in dibenzocyclooctyne 21 (DIBO)121,122 and bicyclo[6.1.0]nonyne 22 (BCN)123, respectively.

The difluorobenzocyclooctyne 23 (DIFBO)124 combined the effect of additional ring strain of the phenyl ring with the effect of fluorine atoms to increase reactivity by the factor of ~1800.

It is so highly reactive that it trimerizes spontaneously, however, it can be stabilized in a complex with cyclodextrine in aqueous solution.

Literature Part – Click Chemistry

Figure 8. Structures of modified cyclooctynes 18–23 and the second-order rate constants

of their reaction with benzyl azide.

Further incorporation of additional sp2-like center or heteroatoms, such as nitrogen or sulfur, very significantly increased the rate of the reaction with azides and also improved the stability of the alkynes (Figure 9). Nitrogen-containing cyclooctynes DIBAC 24125 and BARAC

25126 reacted with azide 4-times and 13-times faster than DIFO-20. It was found, that incorporation of the sulfur atom into a cyclooctyne ring improves the stability but decreases the rate of the cycloaddition with azide.127 The second-order rate constant for cycloaddition of thiacyclooctyne 26 and azide was almost one order of magnitude lower than that of the cyclooctyne 18. Although the rate constant of the thioDIFBO-27 (k = 1.4 × 10–2 M–1 s–1) was almost twenty times lower in comparison with that of DIFBO-23, the sulfur analogue did not oligomerize, thus confirming the stabilizing effect of an endocyclic sulfur atom.

Figure 9. Structures of cycloalkynes 24–29 containing nitrogen or sulfur atom and

the second-order rate constants of their reaction with benzyl azide.

The stability of thiaDIFBO 27 provided an opportunity to enhance the reactivity of the strained sulfur-cycloalkynes (Figure 9). In 1970, the synthesis and properties of stable thiacycloheptyne 28 (TMTH) were reported and it was found that it reacted with phenyl azide and other 1,3-dipoles.128 The second-order rate constant of the reaction with benzyl azide was recently determined to be k = 4.0 M–1 s–1, being the fastest reported cycloalkyne-azide reaction.127 There has been efforts to increase the reactivity of thiocycloheptynes with

Literature Part – Click Chemistry installation of the phenyl ring, however, the highly strained cycloheptyne 29 has never been isolated and it existed only as a transient confirmed by formation of the final triazole.127

Additionally, the biocompatibility of Cu-free click reaction of strained alkynes and azido-triggered biomolecules has been demonstrated in living animals.129,130

1.4.3. Cyclopropenones

The utilization of strained cycloalkynes with azido-triggered biomolecules was complemented by the development of the photochemically triggered click reactions.131

The usage of the light offered an opportunity for spatial and temporal control of labeling of the target substrate. To this end, the photoinduced generation of cycloalkynes from cyclopropenones have been developed (Scheme 7).132,133 The three-membered ring of cyclopropenones 30 is characterized by high thermal stability, and upon irradiation, the release of carbon monoxide and formation of the corresponding alkynes proceeds with quantitative yields.131 This reaction is extremely fast and is complete during the few hundred picoseconds.134 The mechanism of decarbonylation of cyclopropenones 30 starts with the cleavage of one of the carbon-carbon bonds of the cyclopropenone ring, leading to the formation of a zwitterionic structure 31, followed by rapid CO release and production of acetylenes derivative 32 (Scheme 7).

Scheme 7. Mechanism cyclopropenone decarbonylation.

The photochemical generation of the reactive cyclooctynes was first demonstrated by decarbonylation and subsequent Cu-free click reaction of the cyclopropenone 33

(Scheme 8).135 Cyclopropenone 33 did not react with azide in the dark, but efficiently produced the reactive dibenzocyclooctyne 34 upon irradiation. The following cycloaddition with benzyl azide proceeds with the second-order rate constant of k = 7.63 × 10-2 M-1 s-1, which is comparable to that of dibenzocyclooctynol (DIBO-21, Figure 8)121 and, therefore, the aromatic alkoxy-substituents did not influence the rate constant.

Literature Part – Click Chemistry

Scheme 8. Photochemically initiated decarbonylation and the subsequent Cu-free click

reaction of dibenzocyclooctyne and azide.135

The whole process could be easily followed by UV-Vis absorption spectroscopy. The starting cyclopropenone 33 showed two absorption bands at 휆푚푎푥 = 331 and 347 nm. Upon irradiation at 355 nm, decarbonylation and dibenzocyclooctyne 34 formation caused the decrease of these absorption bands and the formation of new bands at 휆푚푎푥 = 300 and 317 nm, corresponding to the formed dibenzocyclooctyne 34. During the reaction with azides, the absorption of cyclooctyne 34 decreased and the corresponding final triazoles 35 were formed.

The biological use of photochemical generation of reactive cycloalkynes, followed by a click reaction with azides was demonstrated in living cells.135 It was shown, that the glycoproteins and glycolipids marked with azido-moiety could be labeled with cyclooctyne 34, photochemically generated in situ from cyclopropenone 33. The Cu-free click reaction of these two species increased the fluorescence intensity of the cell lysates. It was also proved that cyclopropenone 33 did not react with the labeled biomolecules in the dark which was monitored weak fluorescence.

Later, Popik has shown that oxa-dibenzylocyclooctyne 37 can be photochemically generated from the corresponding cyclopropenone ODIBO 36 (Scheme 9).136 The second-order rate constant of alkyne-azide cycloaddition was increased to 2–45 M-1 s-1, which makes the compound 36 suitable for rapid labeling applications in biochemistry.

Scheme 9. Photochemically initiated decarbonylation and the subsequent Cu-free click

reaction of ODIBO 36 and azide.136

Literature Part – Click Chemistry

To increase the rate of photochemically triggered Cu-free click reaction, the more strained cyclopropenone 39 was developed (Figure 10).137 However, the synthesis of dibenzoselenacycloheptyne 40 was not successful; the cyclopropenone 39 was prepared instead, in a similar fashion as in the previous studies.135 Dibenzothiacycloheptyne 41 was found to be unstable138 but the exchange of sulfur for selenium, that is larger than sulfur, should partially decrease the ring strain and instability of cycloheptyne 41.

Figure 10. The structures of dibenzoselena- 39 and 40 and dibenzothiacycloheptyne 41.

The selenocyclopropenone 39 decarbonylated and formed the selenocycloheptyne 42 upon irradiation, which subsequently reacted with benzyl azide in methanol to produce the triazole 43 in quantitative yield (Scheme 10).137

Scheme 10. Photochemical generation of reactive cycloheptyne 42 from cyclopropenone 39

and its subsequent reaction with benzyl azide to give the triazole 43.137

It was also found that when the reaction is performed in THF, a mixture of the target triazole 43 and side product cycloheptene 44.137 The cycloheptyne 42 was not sufficiently stable to be isolated because it abstracted hydrogens from solvents, such as THF and toluene.

This was confirmed by photodecarbonylation of 39 in THF-d8 or toluene-d6 and formation of dideutero-cycloalkene as a single product.

Literature Part – Click Chemistry

Although the reactivity of the photochemically generated cycloheptyne 42 was increased, its utilization in bioorthogonal reactions is limited by formation of its side products.

Literature Part – CORMs

1.5. Carbon Monoxide–Releasing Molecules (CORMs)

Carbon monoxide (CO), typically produced when carbon-containing compounds are only partially burned (incomplete combustion), is a toxic colorless, odorless and tasteless gas and due to these characteristics, CO is called as a “silent killer”. The toxicity for mammals is caused by its much higher affinity to hemoglobin (binding ca. 230-times stronger) than oxygen and the resulting carboxyhemoglobin is not available for transport of oxygen and inhibits oxygen transport to tissues by red blood cell.139,140 The deficiency of oxygen in the tissues, also called hypoxia, and increasing amount of carboxyhemoglobin (up to 50%) causes seizures or coma, in serious cases even death.

Nevertheless, the human body produces a small concentration of CO, the beneficial effects of which were discovered in the 20th century. It was found, that CO is produced during degradation of senescent red cells. The hemoglobin molecules, contained in red cells, are an assemblies of four globular protein subunits (Scheme 11).141 Each subunit is composed from a protein chain closely associated with a non-protein prosthetic heme group 45. The heme group 45 consists of an iron ion which is held in the middle of the heterocyclic porphyrin ring, which is composed from four pyrrole molecules cyclically connected together through the methine bridges.

The rapid degradation of free heme 45 is physiologically important due to its toxicity.142 The degradation involves enzyme heme oxygenase, which cleaves the porphyrin in the presence of NADP and molecular oxygen, forming the carbon monoxide, ferrous ion (Fe2+) and biliverdin (Scheme 11).143 Next degradation step, catalyzed by biliverdin reductase in the presence of NADPH and H+ produces metabolite bilirubin 47, which is then excreted in bile and urine. The amount of CO produced by this route is about 16 μmol h-1 per human body.144

Literature Part – CORMs

Scheme 11. Mechanism of hemoglobin degradation with the generation of CO by heme

oxygenase. Reproduced with permission from Chem. Soc. Rev. 2012, 41, 3571-3583.

CO has been recognized as an important cell signaling molecule145 and a neural messanger,146 and extensive research into the in vivo biology of CO established important functions for CO under various physiological and pathophysiological conditions.147

CO is suggested to have anti-hypertensive, anti-inflammatory and cell-protective effect.147–149

As a gas, CO is freely diffusible and transported through all membranes. Inhalation of CO under controlled conditions reduces pulmonary hypertension,150 presumably by interacting with a smooth muscle signaling proteins, such as guanylyl cyclase. CO inhalation also seems to protect vital organs, such as brain, heart, lung or liver, during ischemia and organ transplantation.151

Moreover, CO inhibits proliferation of the human pancreatic152 and breast cancer cells.153 It has been found that CO influences cellular bioenergetics that is different in the cancer and normal cells.154 CO enhances the mitochondria activity of prostatic and lung cancer cells, resulting in production of reactive oxygen species that trigger metabolic

Literature Part – CORMs exhaustion and cellular collapse causing tumor regression. At the same time, CO inhibits respiration in normal cells and protects them from cell death.

The practical clinical use of CO gas is inhibited by a low solubility of CO gas in water.

Therefore, the distribution of CO to the body fluids and tissues is quite complicated. To reach adequate concentrations of gas in the body, high concentration of CO should be inhaled, which is impossible due to its toxicity. The potential interaction of CO with various molecules in physiological environment also complicates a targeted administration. A strategy to avoid these problems and deliver the controlled amount of CO into the targeted tissues leads to a development of carbon monoxide releasing molecules (CORMs).155 The most important criteria for design of CORMs are their good chemical stability, water solubility and low toxicity for in vivo applications.

CO is known as a ligand in metal complexes for decades, however the therapeutic use as CORMs has been employed only recently.156 Metal-based CORMs can contain essential trace elements (such as manganese, iron and cobalt), as well as non-physiological metals such as ruthenium, iridium or rhenium; selected well-known CORMs are shown in Figure 11.151,157

CORMs can be classified to the three groups due to the mechanism of CO-release: solvent-induced ligand exchange on CORMs, enzyme-triggered CORMs and photoCORMs.

Figure 11. The structures of selected metal-based CO-releasing molecules.157

Literature Part – CORMs

1.5.1. CORMs Based on Solvent-Induced Ligand Exchange

The release of CO is triggered in aqueous media via a solvent-induced ligand exchange. Well-known examples of complexes are the molecules CORM2 48 (complex

[Ru(CO)3(Cl)2]2)156 and CORM3 49 (complex [Ru(CO)3Cl(glycinate)]158) (Figure 11).

The CORM2 48 was one of the first CO-releasing complexes. It is soluble in DMSO and CO is released after ligand exchange with DMSO. The related glycinato complex CORM3 49 provides a better solubility in water and releases CO under physiological conditions.158

The disadvantage of this CORM is exchange of ligands immediately after CORM administration, often not in the targeted place.

1.5.2. Enzyme-Triggered CORMs (ET-CORMs)

Enzymatically triggered CO-releasing molecules are a new and biologically interesting class of CORMs, which have been developed recently.159,160 The compounds, based on acyloxybutadiene tricarbonyl iron species (Figure 11 – ET-CORMs 52 and 53), are activated by enzymes esterases. The esterase cleaves an ester group attached to the acyloxybutadiene

55 (Scheme 12), which generates a highly unstable hydroxybutadiene ligand 56 on the iron center. This instability triggers the decomposition of the molecule via oxidation of [Fe(CO)3], which leads to the release of CO.

Scheme 12. The enzymatically triggered release of CO from acyloxybutadiene tricarbonyl

iron species.160

1.5.3. Photochemically Triggered CORMs (PhotoCORMs)

The development of light-triggered release of CO from a photochemically active

CORM (photoCORMs) is an alternative strategy, which can provide the CO release with a high temporal and spatial resolution in the targeted tissue. The photoCORM molecules can include a metal atom or can be metal-free species. The manganese–based complex 54 with heteroaromatic ligands has been developed as photoCORM (Figure 11), but the release is activated by UV-light (365 nm), which can be harmful to biological tissues and cells.

Literature Part – CORMs

Three organic photoCORMs have been developed in the past years. First visible activated photoCORM is based on cyclic aromatic α-diketone chromophore 58,161 which upon irradiation at 470 nm release two molecules of CO and produces anthracene derivatives 59 as side-product (Scheme 13). Unlike many polycyclic aromatic hydrocarbons, anthracene is not toxic, carcinogenic and mutagenic and the anthracene derivatives also show a low toxicity, which is important for future biological utilization.

Scheme 13. Photoreaction of α-diketone at 470 nm produces two molecules of CO and

anthracene derivatives.161

The fluorescein analogue xanthene-9-acid 60 has been developed as a photoCORM activated by visible light (Scheme 14).162 The carboxylic acid 60 was irradiated at 500 nm in the presence of hemoglobin under physiological conditions. The production of CO was evidenced by formation of carboxyhemoglobin determined by UV-Vis absorption spectroscopy. The proposed mechanism shown in Scheme 14, also studied by isotopically labeled 18O, involves formation of a cyclic lactone 61, which subsequently forms the corresponding ketone 62 while expelling a CO molecule.

Scheme 14. Structure of xanthene-9-acid 60 and proposed mechanism of the CO release.162

Recently, a new generation of transition-metal-free CO releasing molecules based on BODIPY chromophores 63 and 64 (also called COR-BDPs) has been developed

(Scheme 15a).163 These new COR-BDPs were entirely stable under physiological conditions and the GC/RGA analysis showed the release of CO upon irradiation at 500 and 740 nm.

Furthermore, the concentration of released CO was directly proportional to the intensity of incident light, which was established via on-off irradiation and subsequent analysis of CO content. The proposed mechanism starts from the negatively charged BODIPY carboxylate 65

Literature Part – CORMs

(Scheme 15b), which is excited to the singlet state (S1) upon irradiation. It undergoes intersystem crossing (isc) to the triplet state of 66 (T1) followed by photoinduced electron transfer (PET) from the carboxylate to the BODIPY core. The formed biradical 67 undergoes second intersystem crossing to the ground state. The key intermediate, a cyclic α–lactone 68, then releases CO as described previously.162 The proposed mechanism was further supported by DFT calculations.

Scheme 15. a) Structures of new COR-BDPs activated at 500 and 740 nm. b) Proposed

mechanism of CO release.163

1.5.4. Phototherapeutic Window

Near-infrared light can penetrate biological tissues more efficiently than visible light because the tissues scatter and absorb less light at longer wavelength. This is due to biological molecules, such as amino acids, nucleic acids, hemoglobin, bilirubin etc., absorbing light up to 600 nm (Figure 12), whereas water absorbs above ~1 000 nm. A part of the spectrum between 650 and 850 nm is called a tissue–transparent window or phototherapeutic window.164 Therefore, compounds absorbing in this area can be used for photodynamic therapy (PDT),165,166 which is a clinically approved, minimally invasive therapeutic procedure.

In PDT, near-infrared light is used as a source of generation of highly reactive singlet oxygen

(1O2) produced by irradiation of the dyes, such as porphyrins. 1O2 can be generated locally to induce cell apoptosis and death.

Literature Part – CORMs

Figure 12. Absorption spectra of biological molecules and phototherapeutic window

(650-850 nm) characterized by high penetration depth into human tissues. Reproduced with

permission from Coord. Chem Rev. 2016, 325, 67-101. Copyright (2018) American Chemical

Society.

Literature Part – Cyanine Dyes

1.6. Cyanine Dyes

Cyanine dyes are an important class of functional dyes, which are used in many fields of science. The history of cyanine dyes dates back to the middle of 19th century with the first reported synthesis of a blue solid by Williams in 1856.167 Since then, many cyanine dyes and their derivatives have been synthetized. Generally, these dyes are classified into five categories: open chain streptocyanines 69, closed chain cyanines 70, hemicyanines 71, oxonol dyes 72, merocyanine dyes 73 and squaraine dyes 74 (Figure 13).

Figure 13. Classification of cyanine dye derivatives.

1.6.1. Classic Polymethine Cyanine Dyes

Classic cyanine dyes contain two nitrogen-containing heterocycles conjugated through a polymethine chain with a delocalized positive charge (Figure 13, 70). Cyanines are divided to monomethine 75 (Cy1),168 trimethine 76 (Cy3),169 pentamethine 77 (Cy5)170 and heptamethine cyanines 78 and 79 (Cy7)171 according to the number of carbon atoms of the chain (Figure 14). In the recent years, a new structural class of heptamethine cyanines 79 has been introduced, where the polyene region is modified with a heteroatom at C4´ and a carbocyclic ring between C3´ and C5´. This new cyanines are easily synthetized and offer a chance for modification in the meso-position (C4´).172

The cyanine dyes are substances exhibiting various colors and high absorption molar coefficients. The absorption maxima are red-shifted as the length of polymethine chain increases (Figure 15).173 The extension of the chromophore by one vinyl moiety leads to a bathochromic shift of about 100 nm.174

Literature Part – Cyanine Dyes

Figure 14. The structure and absorption maxima of cyanine dyes with different lengths of

the polymethine chain.

Figure 15. The absorption maxima of benzothiazole-based cyanine dyes with different length of polymethine chain. Reproduced with permission from Methods Appl. Fluoresc. 2018, 6,

012001.

Various heterocycles have been used in the cyanine dyes synthesis, such as indole,175 benzothiazole and benzoxazole,175 as well as more conjugated moieties such as quinoline176 or acridine.177

1.6.2. Trimethine and Pentamethine Cyanine Dyes (Cy3 and Cy5)

The absorption maxima of this class of cyanines are generally in the visible region at

500-600 nm for Cy3 and 630-670 nm for Cy5 dyes (76 and 77, respectively, Figures 14 and 15).

The common synthesis of Cy3 and Cy5 dyes involves the condensation of an orthoester with

Literature Part – Cyanine Dyes quaternary heterocyclic salts 80 substituted with an activated methyl group under acidic conditions (Scheme 16).178,179

Scheme 16. The condensation of an orthoester with a heterocyclic salt.

Unsymmetrical Cy3 dyes can be prepared from heterocyclic salts 83 via

Vilsmaeir-Haack formylation and formation of aldehyde 84 as an intermediate (Scheme 17, route a), followed by condensation with the second molecule of heterocycle 85 to produce Cy3 dye 86.180 In a similar vein, N,N-diphenylformamidine 87 or malonaldehyde dianilide hydrochloride 89 are often used in the synthesis of Cy3 and Cy5, respectively (Scheme 17, routes b and c), and subsequent reaction of imines 88 and 90 with second molecule of heterocycle can produce ether symmetrical or unsymmetrical final cyanine dyes 86 and 91.

Scheme 17. Possible synthetic routes for the synthesis of unsymmetrical Cy3 and Cy5 dyes.

Literature Part – Cyanine Dyes

1.6.3. Heptamethine Cyanine Dyes (Cy7)

The Cy7 dyes absorb light in the near IR region, generally between 700 and 900 nm, thus, they are often used in biological applications. The linear Cy7 dyes 93 are commonly prepared by condensation of glucondianil hydrochloride 92 (a commercially available compound or the compound prepared by Zincke reaction; see Chapter 1.6.4), and heterocyclic salts (Scheme 18).181

Scheme 18. Preparation of Cy7 dye from glucondianil hydrochloride.181

The rigid Cy7 dyes containing the 5- or 6-membered cycle 96 in the polymethine chain are prepared by Vilsmeier-Haack formylation of cyclopentanone or cyclohexanone 94, followed by condensation with a heterocycle (Scheme 19).182 The Cy7 substituted with chlorine atom in the meso-position (C4´) 96 of polymethine chain permits further functionalization of these dyes. The meso-chlorine atom can be replaced with appropriate nucleophiles in polar solvents via nucleophilic substitution.172,183 A wide variety of new Cy7 dyes bearing a heteroatom in the meso-position (O, N, S) have been synthetized in the recent years.3,184

Scheme 19. Synthesis of Cy7 dyes from cyclohexanone.

1.6.4. Zincke Reaction

At the beginning of the last century, Zincke has reported a method for ring-opening of the activated pyridinium salts 98 with amines (Scheme 20).185–187 This reaction is now mostly used for synthesis of N-substituted pyridinium salts 100, which are not easily accessible by other means. The iminium salts 102 could also be formed during the reaction and can be used as precursors for preparation of Cy7 dyes.

Literature Part – Cyanine Dyes

The proposed mechanism of Zincke reaction starts with the activation of pyridine 97 via the SNAr reaction with 2,4-dinitrochlorobenzene (Scheme 20).188 The resulting pyridinium salt 98 is known as a Zincke salt. The subsequent ring-opening reaction with primary or secondary amines leads to a cleavage of the pyridinium ring. Nucleophilic attack of primary amines results in a formation of iminium salt 99, which can be recyclized and form a new pyridinium salt 100 (route a). The open structure of intermediate 99 can be attacked with the second molecule of primary amine to form iminium salt 102 subsequently used for Cy7 synthesis (route b).

On the other hand, treatment of pyridinium salts 98 with secondary amines leads to an irreversible ring-opening of pyridinium core and the formation of a stable iminium salt 103

(route c), which can subsequently react with another electrophile. In all cases, the 2,4-dinitroaniline 101 is generated as a side product.

Scheme 20. Zincke reaction ‒ proposed mechanism of ring-opening of Zincke salts with

amines.

Zincke performed the reaction with 2,4-dinitrochlorobenzene. In the following research of König, cyanogen bromide was used for the pyridine activation.189 Today, Zincke reaction and the pyridine activation are important procedures for preparation of heterocycles, such as indoles or pyrrolines, and they are also used in natural product synthesis.190,191

Literature Part – Cyanine Dyes

1.6.5. Photochemistry and Photooxidation of Cyanine Dyes

The cyanine dyes, mainly Cy7 dyes, are often used as fluorescence probes in bioimaging in biological tissues. Among other issues, the use of the cyanine dyes is limited by photooxidation of the cyanine chromophore by singlet oxygen (1O2) produced via energy transfer from the excited triplet state of the dyes.192

The photophysical processes involved in the photooxidation are shown in the

Jabłoński diagram (Figure 16). The dye in the ground state absorbs a photon upon irradiation at a given wavelength and is excited into the first excited singlet state (1S).193,194 The excited singlet state undergoes the intersystem crossing (ISC) to form the triplet excited state. In the presence of oxygen, T–T annihilation can occur. During this process, the triplet state of cyanine dye transfers the energy to the ground-state oxygen (3O2), resulting in the return of cyanine dye to the ground state and formation of a singlet oxygen (1O2) as a reactive species. The singlet oxygen then adds onto the double bounds of the polymethine chain, causing oxidative cleavage of the conjugated system, i.e., photobleaching, to give carbonyl compounds.195

Figure 16. Jabłoński diagram showing the photophysical processes and generation of singlet

oxygen species (1O2).

Schnermann et. al. reported that the photooxidative cleavage of the cyanine dyes 104 occurs predominantly between C2 of the heterocycle and C1´ carbon in 105 and between the C2´- C3´ carbons of the polymethine chain in 106 with yields 71% for 107 and 108 and 10% for 109 and 110, respectively (Scheme 21).195 The oxidative cleavage between the C1´- C2´ and

C3´- C4´ carbons of the polymethine chain has not been observed.

Literature Part – Cyanine Dyes

Scheme 21. Photooxidation of heptamethine cyanine dyes by singlet oxygen (1O2).195

Cyanines can undergo different chemical processes, such as photooxidation,195 aggregation in aqueous solutions,173 photoswitching of the double bonds196 or addition of nucleophiles to the C2 carbon of the heterocycles.197,198 Therefore, new cyanines were developed to improve photostability of these dyes. It was demonstrated that polyfluorinated cyanines 111 (Figure 17) significantly reduced aggregation in aqueous solvents, enhanced the fluorescence quantum yield and decreased the photooxidation with singlet oxygen.199

Similarly, installation of a cyano-group to the position 1 of polymethine chain (Figure 17,

112 and 113) where majority of the singlet oxygen addition occurs, considerably decreases the rate of photoxidation.200,201

Figure 17. Illustration of cyanine dyes with improved photostability.

1.6.6. Utilization of Cyanines

Cyanine dyes are also commonly used as a chemosensors which are defined as chemical systems that convert chemical stimuli into a response that can easily be detected, such as fluorescence, color change or electronic signal. Many reviews have shown that chemosensors can be used as a simple and sensitive detecting tool for variety of analytes, which cannot be detected by traditional methods, especially when they are located within complex environment or biological systems.3,202

For instance, cyanine dyes have been used as pH indicators in biological systems, based on the protonation of the nitrogen-containing ligands presented in the cyanine structure.203–205 It was shown that pH has an important influence in many biological processes

Literature Part – Cyanine Dyes and intracellular pH fluctuation affects cellular behavior, such as cell apoptosis, endocytosis, ion transport through the membranes or muscle contraction.206

In the recent years, sensors based on cyanine dyes for detection of small molecules, such as gasotransmitters (NO,207 H2S208,209), reactive oxygen species (ROS = included peroxides, hydroxyl radicals and singlet oxygen)210,211 and thiols,212,213 have been developed. The cyanine dyes can also be used for releasing of biologically active compounds, such as a gemcitabine used as an anticancer drug.214

Furthermore, cyanine dyes are used as fluorescence probes for enzymes in biological tissues,215 or investigation of tumor cells.216

While photooxidation by singlet oxygen is generally considered as undesirable process, it was recently exploited as a key step in a sequence of reactions leading to the photochemical release of a caged groups upon irradiation with NIR light.217

Results and Discussion – Reverse Micelles

2. Results and Disccusion

2.1. Photoswitching of Azobenzene-Based Reverse Micelles above

and at Subzero Temperatures As Studied by NMR and

Molecular Dynamics Simulations

Results of this project were published in: Filipová L., Kohagen M., Štacko P., Muchová E.,

Slavíček P., Klán P. Langmuir 2017, 33, 2306 – 2317.218 The manuscript is attached in

Appendix 1A.

2.1.1. Introduction

The aim of this work was to propose and synthetize photoresponsive amphiphiles containing an azobenzene moiety. Subsequently, we aimed to prepare reverse micelles and study the behavior of aggregates upon photoisomerization of the azobenzene moiety.

Inspired by previous study of CTAB/water/chloroform-d reverse micelles at room and sub-zero temperatures,14,60 we wanted to study differences in the size and dynamics of the water pool and photoresponsive reverse micelles themselves occurring upon azobenzene E‒Z photoisomerization at both room and sub-zero temperatures.

2.1.2. Results and Discussion

The materials and methods used during the study, as well as NMR spectra of the intermediates and final compound are provided in the Appendix 1B and in its experimental section and supporting information.

2.1.3. Synthesis

The project started with the synthesis of azobenzene-containing amphiphiles performed and optimized during my master study (Scheme 22).219 In the first step, the commercially available p-nitrophenol 114 was alkylated with 1-bromooctane to produce

115. Subsequently, the nitro group was reduced via palladium–catalyzed hydrogenation under the hydrogen atmosphere to give 116. The amine 116 was then used in a classical diazotation reaction with sodium nitrite followed by a coupling reaction with phenol to produce the diazo compound 117. The hydroxy group was then alkylated with excess

1,8-dibromooctane to give 118. In the last step, trimethylamine was used to substitute bromide

Results and Discussion – Reverse Micelles and form the final amphiphile 119 in 59% yield over five steps. In addition, several analogous amphiphiles with different lengths of the alkyl chains were prepared.218,219 Their use in the formation of reverse micelles was not possible due to their low solubility in non-polar solvents.

Scheme 22. The preparation of amphiphile 119.

2.1.4. Photoisomerization of Final Azobenzene-Containing Amphiphile

First, the E‒Z photoisomerization of 119 was studied to establish the ratio of isomers at the photostationary state (PSS). The Z→E isomerization was found to occur both thermally and photochemically. Recently, it has been shown that 4,4´-dialkoxyazobenzenes form thermally stable Z-isomers with lifetimes on the order of hours exhibiting a very slow Z→E isomerization to the thermodynamically more stable E-isomers.89 These facts offer an opportunity to study the amphiphile and its reverse micellar aggregates in the both isomeric forms.

E→Z photoisomerization of 119 was studied using UV-Vis absorption and 1H NMR spectroscopies. The absorption spectrum of E-119 in chloroform shows a strong absorption band at 휆푚푎푥 = 360 nm (Figure 18, blue line). Upon irradiation with UV light (375 nm), E-119 was switched to Z-119 (Figure 18, red line), accompanied by emergence of two absorption bands at 휆푚푎푥 = 310 and 454 nm. The concentration ratio of Z- and E-isomers at the

Results and Discussion – Reverse Micelles photostationary state (PSS) was found to be 96:4, determined by integration of the aromatic ring hydrogen integrals in 1H NMR (Figure 19).

35000 30000 25000 20000 0.60 20000 -1 ~ / cm A

0.45 15000

0.30 10000

0.15 5000 -1 -1  / M cm

 / nm 0.00 0 300 350 400 450 500 550 600 650

Figure 18. UV-Vis absorption spectra of E-119 in chloroform (c = 3 × 10–5 M, blue line) during

irradiation at 375 nm (grey lines) to give the PSS with Z-119 (red line).

The reverse Z→E isomerization of azobenzene moiety can occur both thermally or upon irradiation with visible light. Therefore, the reverse isomerization was studied upon irradiation of Z-119 (96%) at 530 nm to establish the PSS ratio at [E]:[Z] = 95:5, determined by 1H NMR (Figure 19). The spontaneous thermal Z→E isomerization, determined by UV-Vis spectroscopy, was observed with the rate constant of k = (4.96 ± 0.10) × 10–3 s–1 and a half-life

2.3 min. Interestingly, the thermal isomerization of Z-119 reverse micelles, determined by 1H

NMR spectroscopy, was almost two orders of magnitude slower (k = (1.33 ± 0.02) × 10–5 s–1) with a half-life of 14.4 h (vide infra). The forward/reverse photoisomerization was repeated five times to demonstrate that the processes are clean, i.e. without production of any side-products generated during the irradiation.

Results and Discussion – Reverse Micelles

Figure 19. 1H NMR spectra of 119 (c = 25 mM, in CDCl3) during irradiation. i) before irradiation, pure E-119 (marked with blue asterisks), ii) upon irradiation at 375 nm and Z-119 formation (marked with red asterisks), and iii) upon subsequent irradiation at 530 nm.

2.1.5. Reverse Micelles Formation

Followed the previous study of CTAB/chloroform-d/water system,14 the compound

119 was tested to form the reverse micelles in a chloroform-d/water environment.

At the beginning, it was analyzed by 1H NMR spectroscopy, whether 119 in CDCl3 in the presence of water (V = 0.5 mL, c = 100 mM, 10 μL of water) forms reverse micelle aggregates.

The chemical shift of water protons by ~3 ppm indicated that water incorporated inside assembly. The water loading (water incorporated inside an assembly) was determined to be w = ~4. Non-incorporated water was equilibrated on the top of the sample. The micellar properties, such as critical micelle concentration and their photochemical behavior, are described below.

Incorporated water inside an assembly is characterized by the calculated water-to-surfactant molar ratio x and measured water loading w defined as14:

풄(풘풂풕풆풓) 풙 = 풄(풔풖풓풇풂풄풕풂풏풕)

풐풃풔 풄(풘풂풕풆풓 풊풏 풕풉풆 풄풐풓풆) 풘풘풂풕풆풓,풄풐풓풆 = 풄(풔풖풓풇풂풄풕풂풏풕 풎풐풍풆풄풖풍풆풔 풐풇 풕풉풆 풂풔풔풆풎풃풍풚)

If all water molecules are incorporated inside an assembly, then x = w.

Results and Discussion – Reverse Micelles

2.1.6. General Procedure for Preparation of Reverse Micelles

The reverse micelles solutions (c = 50 mM, x = 4) were prepared by direct weighing of

E-119 into the NMR tubes and dissolving in CDCl3 (0.5 mL). The calculated amount of water

(x = 4) was added with a microsyringe, the solutions were vigorously stirred and left to equilibrate for 12 h. Z-RMs were prepared by irradiation of the respective solutions of E-119 at 375 nm until the PSS was reached (~2 h).

2.1.7. Determination of Critical Micelle Concentration

The critical micelle concentration (CMC) is a very important point in the formation of reverse micelles and its determination is based on the measurement of a drastic change of in one of their essential physical properties. Various methods can be applied14,25 (see Chapter

1.2.3.). In this work, 1H NMR spectroscopy was used to study both the CMC and structural changes of assemblies upon irradiation.

To determine the CMC, a set of samples with a different concentration of amphiphile

119 (from 5 up to 250 mM) and a constant concentration of water (x = 4) were prepared directly in the NMR tubes and were allowed to equilibrate for 24 h at 20 °C (selected 1H NMR spectra are shown in Figure 20). The corresponding chemical shifts (훿푤푎푡푒푟) of water protons obtained from 1H NMR, were then plotted as a function of the reciprocal amphiphile´s concentration

(1⁄푐푎푛푎푙푦푡𝑖푐푎푙)(Figure 21). The two straight lines with different slopes were fitted into a graph, and the intersection point corresponds to the critical micelle concentration (CMC) at 푐퐶푀퐶 = 28 ± 0.7 mM. This value is close to the CMC at ~40 mM reported for CTAB/water/chloroform- d system,14 which has the molecular length and geometry comparable to those of E-119.

Results and Discussion – Reverse Micelles

Figure 20. Selected 1H NMR spectra of E-119 in CDCl3/water (x = 4). The signals of water

protons are marked by red asterisks.

푎푛푎푙푦푡𝑖푐푎푙 Figure 21. Dependence of 훿푤푎푡푒푟 on 1⁄푐 for the 119/water/chloroform-d system constructed from the data obtained from 1H NMR shown in Figure 20.

While the chemical shifts of amphiphile remained unchanged with the increasing amphiphile concentration, the chemical shifts of water protons shifted downfield. At a very low concentration (5 mM), the water protons were observed at 1.56 ppm, which is a typical value for water dissolved in chloroform.30 With increasing amphiphile concentration, the downfield chemical shift of water up to 4.00 ppm reflects the formation of hydrogen bonds in growing water pools inside the aggregates.31

Results and Discussion – Reverse Micelles

Based on this measurement, 50 mM solution of 119 was used in the subsequent study.

This concentration is sufficiently above the determined CMC, thus only the reverse micellar aggregates are present in the solution.

표푏푠 The maximum water loading (푤푤푎푡푒푟,푐표푟푒), i.e. the maximum amount of water incorporated inside RMs, was also measured using 1H NMR spectroscopy. A set of samples with a constant concentration of amphiphile 119 (50 mM) and a different loading of water

(calculated as the water-to-surfactant molar ratio x in the range of 0 ≤ x ≤ 15) were prepared.

E-119 was weighed directly into NMR tubes and dissolved in CDCl3. In the case of E-119 RMs, the calculated amount of water was added and the samples were allowed to equilibrate for 24 h at 20 °C. In case of the Z-119 RMs, the prepared samples were first irradiated with UV-light

(375 nm) for 6 h (PSS determined by 1H NMR), then water was added, the samples were allowed to equilibrate for 24 h at 20 °C, and irradiated at 375 nm to preserve the PSS ratio.

Water was incorporated into the structure of RMs until the maximum water loading was reached (Figure 22a and b, for E-119 and Z-119, respectively). The excess of water, which was not incorporated, is phase separated on top of the sample solution. The maximum water

표푏푠 loading 푤푤푎푡푒푟,푐표푟푒 was established to be 4.0 and 2.8 for E-119 and Z-119 reverse micelles, respectively. a) b)

16 10 w obs w obs 14 water core water core 12 8 10 6 8 6 4 4 2 2

0 x 0 x

0 2 4 6 8 10 12 14 16 0 2 4 6 8 10

표푏푠 Figure 22. The plot of 푤푤푎푡푒푟,푐표푟푒 vs. x for the equilibrated a) E-119 and b) Z-119/water/chloroform-d system.

Results and Discussion – Reverse Micelles

2.1.8. Photoisomerization of Reverse Micelles at 303 K

The photochemical behavior of reverse micelles in a 119/water/chloroform-d system was studied by 1H NMR and DOSY techniques at 303 K. The initial solution of reverse micelles was prepared (see Chapter 2.1.6) to study their behavior upon photoisomerization. The initial solution of E-119 RMs was assessed by 1H NMR to show the signal of incorporated water

(Figure 23 and Table 1, Entry 1). The chemical shift of water protons and the water loading indicated that all water molecules are incorporated into the reverse micelles.14,60 The E-119 RM solution was then irradiated at 375 nm in order to produce the Z-119 RMs. After 2 h of irradiation, the PSS was reached containing 96% of the Z-isomer. At this point, changes in the parameters were compared with those of the initial state (Table 1, entry 2). The chemical shift of water protons moved upfield from 2.86 ppm to ~2.3 ppm, and water loading decreased from 4.0 to 2.7. The parameters of the water protons indicated that the size of the reverse micelles decreased upon E→Z isomerization, however, RMs were not completely destroyed.

A new signal in 1H NMR at ~4.6 ppm was observed (Figure 23, entry 1a) and assigned to bulk water. This value corresponds to the amount of water released from the core. This released water eventually equilibrated as a top layer on the chloroform solution and was not visible in the 1H NMR spectra anymore (Figure 23, entry 2).

The diffusion coefficient of E-119 RMs in the initial solution was determined to be

D = (3.9 ± 0.4) × 1010 m2 s–1 with a hydrodynamic radius of Rh = 1.11 ± 0.12 nm (Table 1, entry 1).

After 2 h of irradiation at 375 nm, Z-119 RMs were formed. The corresponding diffusion coefficient was found to be D = (4.5 ± 0.4) × 1010 m2 s–1 with a hydrodynamic radius of

Rh = 0.96 ± 0.09 nm (Table 1, entry 2). The diffusion coefficients did not change significantly, and the corresponding hydrodynamic radii only slightly decreased. Moreover, the D values were compared with the diffusion coefficient for linear premicellar aggregates (c = 10 mM, x = 4) and were found to be D = 6.6 × 10−10 s−1. The difference in the diffusion coefficient values demonstrated, that the reverse micelles stayed retained upon irradiation; the reverse micelles were not destroyed, only the size was partially reduced.

Results and Discussion – Reverse Micelles

Figure 23. Selected 1H NMR spectra of 119/water/chloroform-d reverse micelles during irradiation at 303 K. 1) Initial solution (pure E-RMs); 1a) RMs upon irradiation at 375 nm for 2 h; 2) RMs upon irradiation at 375 nm for 10 h; 3) RMs upon subsequent irradiation at 530 nm; 4) RMs without irradiation, thermal isomerization occurred. Signals for E-119 are marked with green asterisks, those of Z-119 with black asterisks, signals of water protons in the water core are marked with red asterisks and those of the bulk water are assigned with blue asterisk.

Table 1. Irradiation of 119/water/chloroform-d reverse micelles at 303 K.

풐풃풔 10 2 -1 entry 흀풊풓풓 / nm [E-119]/[Z-119] 휹풘풂풕풆풓,풄풐풓풆 / ppm 풘풘풂풕풆풓,풄풐풓풆 D /10 m s 푹풉 / nm 1 Dark 100 : 0 2.86 ± 0.03 3.95 ± 0.07 3.9 ± 0.4 1.11 ± 0.12 2 375 4 : 96 2.31 ± 0.06 2.73 ± 0.07 4.5 ± 0.4 0.96 ± 0.09 3 530 95 : 5 2.40 ± 0.07 2.81 ± 0.09 4.4 ± 0.3 0.99 ± 0.07 4 Dark 99: 1 2.67 ± 0.04 3.75 ± 0.05 4.1 ± 0.03 1.07 ± 0.08

The Z-119 RMs were irradiated at 530 nm and the Z-119 switched back to E-119.

A complete photoisomerization was reached after 1 h with the PSS of [E-119]/[Z-119] = 95:5

(Figure 23 and Table 1, entry 3). The chemical shift as well as the water loading remained virtually unchanged, which was confirmed by the value of the diffusion coefficient and

Results and Discussion – Reverse Micelles the corresponding hydrodynamic radii determined by NMR. This demonstrated that Z-RMs are switched to E-RMs without any influence on the structure of reverse micelles under the investigated conditions.

However, when the sample was left to stand in the dark for 10 h, the values returned to approximately the same values corresponding to those of the initial sample (Figure 23 and

Table 1, entry 4). The separated water was incorporated back to the water core, which was indicated by the increased water loading w and diffusion coefficient D. The phase-separated water incorporation into the reverse micelles was also previously observed in the CTAB RMs study with a rate constant of 8.1 × 10-6 s-1.60

As demonstrated before, the Z-119 RMs are smaller than the E-119 RMs (Figure 23,

표푏푠 Table 1). Therefore, E-119 RMs with 푤푤푎푡푒푟,푐표푟푒 ~ 2.8, corresponding to the maximum water loading for Z-119 RMs, were prepared. The E-119 RMs were irradiated at 375 nm followed by irradiation at 530 nm, and 1H NMR spectra were measured after every irradiation step. All the characteristics, such as chemical shift of water protons (훿푤푎푡푒푟,푐표푟푒) or water loading 표푏푠 (푤푤푎푡푒푟,푐표푟푒) remained unchanged, and no water shedding was observed (Table 2). Based on these experiments, we could conclude that E–Z isomerization, as well as the backwards Z–

E isomerization did not influence the properties of RMs, and thus the size of the reverse micelles remained intact.

Table 2. Irradiation of smaller micelles (c = 50 mM, x = 2.8).

풐풃풔 entry 흀풊풓풓 / nm [E-119]/[Z-119] 휹풘풂풕풆풓,풄풐풓풆 / ppm 풘풘풂풕풆풓,풄풐풓풆 1 Dark 100 : 0 2.31 2.86

2 375 4 : 96 2.14 2.84 3 530 93 : 7 2.24 2.83

Contrary to some photoresponsive systems of azobenzene-containing aggregates,92,98,100 the 119/water/chloroform-d system shows exceptional stability during photoisomerization. Only a small fluctuation of some parameters, such as the chemical shift

표푏푠 of water protons (훿푤푎푡푒푟,푐표푟푒), water loading (푤푤푎푡푒푟,푐표푟푒), diffusion coefficient D or hydrodynamic radii Rh was observed. A small upfield shift of 훿푤푎푡푒푟,푐표푟푒 and also a small decrease in Rh demonstrated a partial water shedding but not the disruption of reverse

Results and Discussion – Reverse Micelles

micelles. In addition, the 훿푤푎푡푒푟,푐표푟푒 above ~2.2 ppm is characteristic for water incorporated inside assemblies and above the value typical for premicellar aggregates (1.8 ppm).14

In the same fashion, the diffusion coefficients D and corresponding Rh are larger than those of linear premicellar aggregates.

We conclude that the RM system is quite stable during photoisomerization, especially

표푏푠 in the case of smaller RMs (푤푤푎푡푒푟,푐표푟푒 = 2.8). The partial water shedding can be caused by different packing, micellar size and different resulting surface properties, such as different arrangement of the amphiphile chains.

2.1.9. Molecular Dynamic Simulations (work of E. Muchová and P. Slavíček)

The methods of molecular dynamics (MD) can provide the information about the physical movement of atoms and molecules and give detailed information about the microscopic structure of RMs that are not accessible experimentally. Here, molecular dynamic simulation, performed by E. Muchová and P. Slavíček, was used to describe the structure of RMs and study the stability of both isomeric forms.

According to the calculation results (Figure 24), the water molecules form a compact, more or less spherical, droplet with the diameter of ~1 nm, which is in agreement with the experimentally determined value of Rh of the micelle (see Table 1). Azobenzene moiety were found to undergo E-Z photoisomerization and reorganization in space without any significant influence of the RMs structures. The structural characterization of 119/water/chloroform-d micelles is also consistent with the previous studies on CTAB reverse micelles.60

Results and Discussion – Reverse Micelles

Figure 24. Representative snapshots of E- and Z-RMs at left and right, respectively. Bromide ions are colored in pink, oxygen atom in red, hydrogen atoms in white, carbon atoms in cyan and nitrogen atoms in blue. MD simulations was performed by E. Muchová and P. Slavíček.

2.1.10. Reverse Micelles at Subzero Temperatures

The behavior of RMs at subzero temperatures was studied in the range of 303–233 K for both E- and Z, isomers. RM solutions (푐푎푛푎푙푦푡𝑖푐푎푙 = 50 mM, x = 4) in the both isomeric forms were prepared (see Chapter 2.1.8.; Table 1, entries 1 and 4 for initial solution of E- and Z-RMs, respectively). The RM solutions were then cooled to 253 or 233 K in a cooling bath and left to equilibrate for 1 h. The frozen samples were inserted into a precooled NMR spectrometer, and

1H NMR spectra at the corresponding temperature were recorded. At 253 as well as 233 K, the water signal disappeared in both isomeric forms of RMs, which did not allow the calculation

표푏푠 of 푤푤푎푡푒푟,푐표푟푒. Subsequently, the frozen samples were warmed back to 303 K, and their physico-chemical properties were studied.

In the case of E-119 RMs, the subzero temperatures caused precipitation of 119; only 30 and 27% stayed in the solution at 253 and 233 K, respectively. Upon a rapid warming the sample to 303 K, the precipitate immediately dissolved and the initial concentration was

표푏푠 restored. The values of 훿푤푎푡푒푟,푐표푟푒 and 푤푤푎푡푒푟,푐표푟푒 decreased only by about 10 and 5% for 253 and 233 K, respectively (Table 3, entries 2 and 3), and only a very small water loading was observed. The size of RMs did not change after the freeze–thaw cycle. The retention of the

RMs was caused by precipitation of the entire aggregates from the solution, thus preventing water shedding. A higher viscosity of chloroform at 233 K might also contribute to the stabilization of the RMs. All these data demonstrated, that E-119 RMs are retained at subzero temperatures. These results are in agreement with the study of reverse micelles composed of

CTAB/water/chloroform-d system after freezing to 233 K and subsequent thawing.60 58

Results and Discussion – Reverse Micelles

Table 3. Changes in the RMs properties induced by freezing. All data were measured at

303 K.

풐풃풔 10 2 -1 Entry 119 T/K 휹풘풂풕풆풓,풄풐풓풆 / ppm 풘풘풂풕풆풓,풄풐풓풆 D /10 m s 푹풉 / nm c(119)/mM 1 [E-119] 303 2.86 ± 0.03 3.95 ± 0.07 3.9 ± 0.4 1.11 ± 0.12 50 2 253 2.68 ± 0.11 3.48 ± 0.10 4.5 ± 0.2 0.96 ± 0.04 15 3 233 2.81 ± 0.09 3.79 ± 0.09 4.0 ± 0.1 1.08 ± 0.03 13

4 [Z-119] 303 2.31 ± 0.06 2.73 ± 0.07 4.5 ± 0.4 0.96 ± 0.09 50 5 253 2.12 ± 0.12 2.11 ± 0.11 4.7 ± 0.4 0.92 ± 0.08 50 6 233 2.21 ± 0.07 2.36 ± 0.08 4.8 ± 0.4 0.94 ± 0.08 50

Contrary to E-119 RMs, Z-119 RMs did not precipitate and retained the initial concentration even at 253 and 233 K (Table 3, entries 5 and 6). The freezing of Z-119 RMs

표푏푠 induced a greater water shedding because 푤푤푎푡푒푟,푐표푟푒 decreased from 2.73 ± 0.07 to 2.11 ± 0.11 upon freezing to 253 K and subsequent thawing. This corresponds to 33% of the initial water

표푏푠 loading 푤푤푎푡푒푟,푐표푟푒. Interestingly, water shedding is suppressed by freezing to lower 표푏푠 temperature (233 K) where Z-119 RMs expelled only 15% of water (푤푤푎푡푒푟,푐표푟푒 decreased to 2.36 ± 0.07). The same trend was observed at a low temperature study of the

CTAB/water/chloroform-d system.60

2.1.11. Photoisomerization of Reverse Micelles at Subzero Temperatures

The photoisomerization of photoresponsive reverse micelles was studied also at subzero temperatures (253 and 233 K). First, the equilibrated solutions of E-119 RMs and

Z-119 RMs (Table 4, entries 1 and 4 for E-119 RMs and Z-119 RMs, respectively) were cooled to ambient temperature (253 or 233 K) in a cooling bath and were left to equilibrate for 1 h.

The samples were then irradiated at those temperatures with 375 or 530 nm in the cases of

E-119 and Z-119 RMs, respectively. When the PSS was reached, the samples were inserted into a precooled NMR spectrometer and 1H NMR spectra were recorded. Afterwards, the samples were quickly warmed up to 303 K and 1H NMR spectra were recorded again.

Because precipitation occurred at subzero temperatures (see Chapter 2.1.10. Table 3) in the case E-119 RMs, the photochemical experiment started with the precipitated sample. After irradiation at 375 nm, [E-119 RMs]/[Z-119 RMs] was found to be ~3:97 at both 253 and 233 K.

However, after warming samples to 303 K, the precipitate dissolved and the final ratio was

Results and Discussion – Reverse Micelles established to be 7:93 and 70:30 for 253 and 233 K, respectively (Table 4, entries 2 and 3). A lower efficacy of photoisomerization at 233 K might be influenced by the precipitation of E-

119 RMs at 233 K, which may act as an internal optical filter, decreasing the final ratio. In addition, the steric constraints in rigid environment might increase the barrier for E→Z photoisomerization.

Table 4. The changes in the RMs properties upon photoisomerization performed at subzero temperatures.

풐풃풔 10 2 -1 entry T/K [E-119]/[Z-119] 휹풘풂풕풆풓,풄풐풓풆 / ppm 풘풘풂풕풆풓,풄풐풓풆 D /10 m s 푹풉 / nm c(119)/mM 1 303 Only [E-119] 2.87 ± 0.07 3.95 ± 0.07 3.9 ± 0.4 1.11 ± 0.12 50 2 253 7 : 93 2.42 ± 0.10 2.68 ± 0.09 3.9 ± 0.2 1.11 ± 0.06 50 3 233 70 : 30 2.60 ± 0.08 3.45 ± 0.05 4.2 ± 0.2 1.03 ± 0.05 13 4 303 4 : 96 2.31 ± 0.06 2.73 ± 0.07 4.5 ± 0.4 0.96 ± 0.09 50 5 253 95 : 5 2.27 ± 0.07 2.59 ± 0.10 4.0 ± 0.2 1.08 ± 0.06 1.5 6 233 95: 5 2.35 ± 0.04 2.51 ± 0.08 4.1 ± 0.2 1.06 ± 0.05 n.d.

Likewise, Z-119 RMs were cooled to subzero temperature and irradiated at 530 nm.

The behavior of aggregates was opposite compared to that of E-119 RMs. While Z-119 RMs were fully dissolved at the subzero temperatures, increasing formation of the precipitate was observed during irradiation at 530 nm and only imperceptible amount stayed in the solution:

3 and <1% at 253 and 233 K, respectively (Table 4, entries 5 and 6). Subsequently, the samples were warmed to 303 K and the precipitate immediately dissolved to recover the initial concentration. Nearly a complete conversion to E-119 RMs with the ratio

[E-119 RMs]/[Z-119 RMs] = 95:5 at both 253 and 233 K was observed. All the characteristics of micelles remained unchanged, demonstrating that Z→E photoisomerization occurred at subzero temperatures without any influence on the structure of aggregates.

In order to investigate the effect of the varying amphiphile configuration on the solubility of RMs, Z→E photoisomerization (530 nm at 233 K) was followed by 1H NMR spectroscopy (Figure 25). Majority of the micelles stayed in the solution (illustrated as blue triangles), until the increasing concentration of E-119 RMs and decreasing concentration of Z-119 RMs (red squares and black circles, respectively) reached the ratio of ~1:1. At this point, the total concentration of 119 drastically decreased, which was observed

Results and Discussion – Reverse Micelles as a precipitation of the RMs. These results are in an agreement with the observation of E-119

RMs precipitating at subzero temperatures (see Chapter 2.1.10.).

Figure 25. Dependence of the concentration of 119 on the time of irradiation of the Z-119 RMs at 233 K at 530 nm. Total concentration of both E-119 and Z-119 RMs (blue triangles) and the concentration of Z-119 RMs (black circles) and E-119 RMs (red squares) is depicted.

2.1.12. Conclusions

A new azobenzene-containing amphiphilic molecule was synthetized, and it was shown that the 119/water/chloroform-d system forms stable reverse micelles with the critical micelle concentration of ~28 mM. It has been demonstrated that reverse micelles retained upon photoisomerization, and only a partial water shedding was observed due to the smaller water pool in the Z-119 RMs. The photoisomerization occurred also at subzero temperatures with the retention of RMs. It was shown, that E-119 RMs are less soluble at lower temperatures and precipitated from the solution. The retention of RMs was also supported by molecular dynamic simulations, performed by E. Muchová and P. Slavíček, who demonstrated the structures of RMs in the case of both E-119 and Z-119 RMs.

2.1.13. Author´s Contribution

P. Štacko designed the target molecule, L. Filipová synthetized the compounds and performed all NMR and spectroscopic measurements at room and subzero temperatures. E. Muchová and P. Slavíček performed the molecular dynamics simulations.

Results and Discussion – Silacyclopropenone

2.2. Photochemical Formation of Dibenzosilacyclohept-4-yne for

Cu-Free Click Chemistry with Azides and 1,2,4,5-Tetrazines

Results of this project were published in: Martínek M., Filipová L., Galeta J., Ludvíková

L., Klán P. Org. Lett. 2016, 18, 4892 – 4895.220 The manuscript and its supporting informations are attached in Appendices 2A and 2B.

2.2.1. Introduction

The past decade has seen rapid development of click chemistry.221,222 This synthetic approach is used for connection of two molecular building blocks in a reaction, which is fast, high yielding, selective, easy to perform, under mild conditions, and with few or no byproducts.102,223 The recent development of Cu-free click reaction does not require a cytotoxic metal catalyst and offer a unique method for labelling of biomolecules in living cells.224 One of the common bioorthogonal reactions includes the 1,3–dipolar cycloaddition of strained alkynes to the azides, also called strain-promoted cycloaddition.118,120

The development of new strained alkynes and improvement of their reactivity is one of the aims of current research.133,136,137

The goal of this study was to synthetize the silacyclopropenone 120, which should photochemically release carbon monoxide (CO) to produce the reactive strained cycloheptyne

121 (Scheme 23). The project was inspired by work of Popik135 and Bertozzi137 who developed the photochemical generation of strained alkynes from cyclopropenones 33 and 39, respectively (see Chapter 1.4.3). The molecule 120 was designed according an idea to increase the ring strain of photochemically generated cycloheptyne 121 to speed up the subsequent reaction with azides. Inspired by the work on strained selenocycloheptyne 42,137 we aimed to exchange selenium by the silicon atom, which is larger than selenium (Van der Waals radius are 210 and 190 pm for Si and Se, respectively).225 We hoped that the incorporation of silicon into the strained cycloheptyne structure improves its stability, suppresses the side reactions, and simultaneously, maintaines ring strain to induce a fast Cu-free click reaction.

Results and Discussion – Silacyclopropenone

Scheme 23. The photochemical generation of strained cycloheptyne 121.

2.2.2. Synthesis (work of J. Galeta)

J. Galeta synthetized the target silacyclopropenone 120 in the two steps synthesis, which included lithium‒halogen exchange of 3-bromoanisole 122 with n-butyllithium, followed by the reaction of aryl lithium with dichlorodimethylsilane to produce the intermediate 123 (Scheme 24). In the second step, two consecutive Fridel-Crafts alkylations of 123 with tetrachlorocyclopropene catalyzed by aluminium chloride as a Lewis acid and subsequent hydrolysis provided the target cyclopropenone 120 in overall 30% yield.

Scheme 24. Synthesis of cyclopropenone 120.

2.2.3. Photochemistry of Silacyclopropenone

The absorption spectrum of cyclopropenone 120 in methanol displays two close-lying

4 –1 3 –1 absorption bands at 337 and 354 nm with 휀354 ≈ 2.6 × 10 mol dm cm (휆푚푎푥 = 336 and 353

4 –1 3 –1 nm, 휀353 ≈ 1.8 × 10 mol dm cm in acetonitrile), comparable to the structurally similar bicyclo[6.1.0]nonatrien-9-one derivative135 (Figure 26, black line). In methanol, the cyclopropenone 120 is only weekly fluorescent with 휆푒푚 = 387 nm with the quantum yield of fluorescence below 0.01 (Figure 26, red line).

Results and Discussion – Silacyclopropenone

4x104 3x104 2x104 30000 1.0  / cm-1 25000 0.8

20000

0.6 15000

0.4

10000

-1

5000 0.2

emission / a.u. emission -1

/ M  / nm 0  0.0 250 300 350 400 450 500

Figure 26. Absorption and emission spectra of cyclopropenone 120 in MeOH (black and red

line, respectively).

2.2.4. Irradiation of Silacyclopropenone in the Presence of Benzyl Azide

To study the click reaction of the photochemically generated cycloheptyne 121 with benzyl azide, the solution of cyclopropenone 120 and benzyl azide was irradiated (375 nm) in methanol or acetonitrile, and the triazole 124 was isolated as a single product in very good yields (93% and 97%, respectively; Scheme 25).

Scheme 25. Photochemical generation of cyclooctyne 121 and the subsequent click reaction

with benzyl azide.

The photochemical generation of silacycloheptyne 121 and subsequent Cu-free click reaction was studied by UV-Vis and 1H NMR spectroscopy (Figures 27 and 28, respectively).

Upon irradiation at 375 nm in methanol, the absorption bands at 휆푚푎푥 = 337 and 354 nm decreased with a concomitant formation of two new bands at 휆푚푎푥 = 315 and 334 nm, shown in Figure 27 as a red line. The generated absorption spectrum corresponds to the silacycloheptyne 121. The quantum yield of photodecarbonylation Φ = 0.58 ± 0.01 is comparable to the published cyclooctyne derivatives.135 In the presence of benzyl azide, the absorption bands of silacycloheptyne 121 decreased due to cycloaddition with benzyl azide

Results and Discussion – Silacyclopropenone and formation of the final triazole 124 absorbing at 283 nm (Figure 27, blue line). The photochemistry of cyclopropenone 120 had the same behavior in acetonitrile with absorption bands at 휆푚푎푥 = 316 and 355 nm and quantum yield of photodecarbonylation Φ = 0.71 ± 0.01.

Figure 27. Irradiation of cyclopropenone 120 (c = 4.5 × 10–5 M) in the presence of benzyl azide

(c = 4.5 × 10–5 M) in methanol followed by UV-Vis spectroscopy. Initial solution of 120 and

BnN3 (black line), the intermediate 121 (red line) and the product 124 (blue line).

After 1 h of irradiation in methanol-d4, clear emergence of tetrazine 124 is visible

(Figure 28). After 2 h of irradiation, the conversion of cyclopropenone 120 was complete and the exclusive formation of the target tetrazine 124 as a single product is observed.

Results and Discussion – Silacyclopropenone

iv) 2 h at 375 nm

iii) 1 h at 375 nm

ii) Cyclopropenone 120 + BnN3

i) Cyclopropenone 120

Figure 28. Irradiation of cyclopropenone 120 (4.8 mg, 0.015 mmol) in the presence of benzyl azide (20μL of 1M solution in CD3OD-d4, 0.020 mmol) in CD3OD-d4 followed by 1H NMR.

Cyclopropenone 120 (entry i) and initial solution of 120 and BnN3 (entry ii). The mixture at

1 and 2 h of irradiation at 375 nm (entries iii and iv, respectively).

It was observed that the cycloheptyne 121 slowly photochemically decomposes upon irradiation at 375 nm, however, the photodegradation is several orders of magnitude slower than the corresponding cycloaddition and it does not interfere with the click reaction.

Upon extensive irradiation of cyclopropenone 120 (c = 4.5 × 10–5 M) in methanol for 72 h, an orange precipitate was formed. 1H, 13C, 2D NMR and HRMS experiments suggested that the solid corresponds to oligomeric compounds consisting of at least two parent subunits. The products of alkyne trimerization124 or aryne insertion into the carbonyl bond226 were not observed in this case. Efficient hydrogen transfer from H-atom-donating compounds or solvents to some cycloheptynes has been reported before.137 In the case of silacycloheptyne

121, no products of hydrogen transfer from the solvent, benzyl azide or even from benzyl alcohol (20 eq.) added to the reaction haven been identified.

The reaction kinetics was also studied by transient spectroscopy performed by

L. Ludvíková. She found that the singlet excited state of cyclopropenone 120 with a lifetime

Results and Discussion – Silacyclopropenone of (1.50 ± 0.03) ps is formed immediately after excitation. This state very quickly converts to the singlet excited state of cycloheptyne 121 with a lifetime of (28.99 ± 0.39) ps, which subsequently converts to the triplet excited state with a lifetime exceeding 1.5 ns, which is the longest time delay of our pump-probe setup.

2.2.5. Cu-free Click Reaction of Silacyclopropenone with Tetrazines

Tetrazines are another type of molecules feasible for bioorthogonal reactions227 and they are known for their very fast [4+2] cycloaddition reaction with strained alkynes and alkenes.228 Reaction of the cyclopropenone 120 with several commercially available tetrazines was performed in the same manner as in the case of benzyl azide (Scheme 26).

The corresponding pyridazine derivatives were obtained via an inverse-electron demanding

Diels-Alder reaction with the photochemically generated 121 in very good yields (Table 5).

The pyridazine derivatives 126b and 126c were prepared in 92% and 93% in acetonitrile and methanol, respectively. However, the pyridazine 126a was prepared in chloroform due to its low solubility in the previously used polar solvents.

Scheme 26. Photochemically initiated reaction of the cyclopropenone 120 with tetrazines

125a-c.

According to the literature, inverse-electron-demand Diels-Alder reaction between the tetrazines and strained alkynes is faster than the cycloaddition with benzyl azide

(second-order rate constants are 1–104 M–1 s–1 and 10-2–1 M–1 s–1 for tetrazines and azides, respectively).227,229 The same trend was observed for 120, where the second-order rate constant for the reaction with the tetrazine derivative 125b was one order of magnitude faster than the reaction with benzyl azide (Table 5).

Results and Discussion – Silacyclopropenone

Table 5. Summarized data of photochemically initiated Cu-free click reactions between 120 or tetrazines 125a-c and BnN3.

Compound Producta Rate constant of reaction / M–1 s–1 b

BnN3 124 (93%, MeOH) (22.5 ± 0.7) in MeOH, (15.8 ± 0.9) in ACN

Tetrazine 125a 126a (84% in CHCl3) Tetrazine 125b 126b (92% in ACN) (2.58 ± 0.15) × 102 in ACN Tetrazine 125c 126c (93% in MeOH)

aProducts and isolated yields. bSecond-order rate constant of click reaction between 121 and

BnN3 or tetrazines 125a-c.

The reactivity of two previously published cyclopropenones 33 and 39 has been reported only with azides (see Chapter 1.4.3.).135,137 Therefore, their reactivity with tetrazine

125a was investigated.

The both cyclopropenones 127 and 39 (Figure 29) were synthetized according to the published procedures.135,137,230 In cyclopropenone 127, the –OMe were installed instead of the original –OBu groups in 33, which did not influence the physico–chemical properties of the cyclopropenone.

In a reaction of tetrazine 125a with cyclopropenone 127 (Figure 29), no formation of the target pyridazines was identified in the dark. While the cyclooctyne 128 formation was observed upon irradiation at 375 nm, it did not react in the subsequent cycloaddition even at a higher temperature (60 °C). Similar cyclooctyne 34 was reported to be sufficiently stable to be isolated,135 therefore, we concluded that 128 is too stable to react with tetrazines. In similar fashion, selenocyclopropenone 39 in the presence of tetrazine 125a also did not provide any target pyridazine upon irradiation. Moreover, the mixture of unidentified products was obtained possibly due to the previously reported side reactions, such as hydrogen abstraction from solvents.137

Figure 29. Previously published cyclopropenones (127, 39) and the corresponding

cycloalkynes (128, 42) 68

Results and Discussion – Silacyclopropenone

2.2.6. Conclusion

Herein the synthesis of stable silacyclopropenone 120 and photochemical generation of the strained silacycloheptyne 121 was shown. The resulting cycloheptyne 121 undergoes a rapid and selective Cu-free click reaction with azides and tetrazines. The second-order rate constant of 121 with benzyl azide (22.5 M-1 s-1) was two order of magnitude faster than that of the corresponding reaction of previously published dibenzocyclooctyne 34 (7.6 × 10-2 M-1 s-1), faster than that of the thiacycloheptyne derivative 28 (4.0 M–1 s–1) and comparable to the oxabenzocyclooctynes 37 (2 – 45 M-1 s-1).136 The silacycloheptyne 121 is thus one of the fastest reported Cu-free cycloalkyne–azide click reaction with the rate constant comparable to those for common Cu catalyzed alkyne–azide cycloadditions.227 Moreover, the second-order rate constant of silacycloheptyne 121 with tetrazines is around one order of magnitude faster

(2.58 × 102 M-1 s-1) than that of for benzyl azide (15.8 M-1 s-1).

2.2.7. Author´s Contribution

M. Martínek and L. Filipová performed the photochemical experiments of decarbonylation and subsequent click reaction. M. Martínek and L. Ludvíková studied the transient-absorption spectroscopy of the photochemical process. J. Galeta synthetized the targeted cyclopropenone 120.

Results and Discussion - Cyanine Dyes

2.3. Development of New PhotoCORMs based on Heptamethine

Cyanine Dyes

Confidental content

Access is restricted until 1.1.2021

Results and Discussion - Cyanine Dyes

2.4. Cyanine Dyes Substituted at the Heptamethine Chain Accessed

by Ring-Opening of Pyridnium Salts

Confidental content

Access is restricted until 1.1.2021

Experimental Part

3. Experimental Part

3.1. Materials and Methods

Reagents and solvents of the highest purity available were used as purchased, or they were purified/dried using the standard methods when necessary. Synthetic procedures were performed under ambient atmosphere unless stated otherwise.

Flash column chromatography was performed using silica gel (230−400 mesh).

1H NMR spectra were recorded on a Bruker Avance II 300 MHz or on a Bruker Avance III 500

MHz spectrometers, and those of 13C NMR were obtained on 125 MHz or 75 MHz instruments in CDCl3, CD3OD, DMSO-d6 and D2O. 1H chemical shifts are reported in ppm relative to tetramethylsilane ( = 0.00 ppm) using the residual solvent signal as an internal reference.

13C chemical shifts are reported in ppm with CDCl3 ( = 77.23 ppm), CD3OD ( = 49.00 ppm) and DMSO-d6 ( = 39.50 ppm) as internal references. The deuterated solvents were kept under nitrogen atmosphere.

UV-vis spectra were obtained on an Agilent 8453 UV-Vis spectrophotometer with matched 1.0 cm quartz cuvettes. Fluorescence was measured on an automated luminescence spectrometer in 1.0 cm quartz fluorescence cuvettes at 26 ± 1 °C. The corresponding optical filters were used to avoid the second harmonic excitation/emission bands induced by the grating. The samples of concentrations with the absorbance below 0.1 at the excitation wavelength at absorption maxima were used. Each sample was measured five times and the spectra were averaged. Emission and excitation spectra are normalized and smoothed using standard protocols.

The exact masses of the compounds were obtained by a triple quadrupole electrospray ionization mass spectrometer in a positive or negative mode coupled with direct inlet or liquid chromatography.

Melting points were measured on a non-calibrated Kofler’s melting point apparatus with samples placed between two glass plates.

NMR spectra are attached in Appendix C.

Experimental Part

3.2. Synthesis

Confidental content

Access is restricted until 1.1.2021

Conclusion

4. Conclusion

This dissertation thesis discusses three separate projects.

In the first one, a photoresponsive amphiphiple containing the azobenzene moiety was synthesized and characterized. It was subsequently used for formation of reverse micelles in a chloroform-d/water system with a critical micelle concentration of ~28 mM. The behavior of the aggregates was studied at room and subzero temperatures. It was demonstrated that azobenzene-containing amphiphile forms stable reverse micelles, which are, contrary to other similar systems, retained even upon photoisomerization of the azobenzene moiety under various conditions.

In the second project, a new stable silacyclopropenone was developed. It was shown that silacyclopropenone undergoes quantitative photochemical decarbonylation with a concurrent formation of a strained alkyne. The corresponding silacycloheptyne reacted with benzyl azide and various tetrazines in a Cu-free click reaction. The resulting triazole and pyridazines were isolated as single products in high yields (up to 84%). The second-order rate constant were determined and it was found that the reaction with tetrazine is one order of magnitude faster than the reaction with azide. This reaction was found to be one of the fastest reactions of strained alkyne in a Cu-free click process reported to date.

The last project was focused on the synthesis of new heptamethine cyanine dyes with the prospect of developing new photoCORMs. Differently substituted pyridinium salts were used in a pyridinium ring-opening reaction, and the following condensation with activated heterocycles provided the target cyanine dyes. The reaction conditions were optimized to provide the cyanines under mild conditions and with high yields. Thus, a series of new Cy7 dyes with different functional groups installed on the heptamethine chain was prepared for the first time.

112

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List of Abbreviations

6. List of Abbreviations

ACN acetonitrile

AOT aerosol OT (= sodium bis(2-ethylhexyl)sulfosuccinate)

CMC critical micelle concentration

CO carbon monoxide

COR-BDPs carbon monoxide-releasing molecules based on BODIPY

CORMs carbon monoxide-releasing molecules

CTAB cetyl trimethylammonium bromide

CTAC cetyl trimethylammonium chloride

CuAAC copper-catalyzed alkyne-azide cycloaddition

Cy3 trimethine cyanine dye

Cy5 pentamethine cyanine dye

Cy7 heptamethine cyanine dye

D diffusion coefficient

DCC N,N´-dicyclohexylcarbodiimide

DCM dichloromethane

DLS dynamic light scattering

DMF dimethylformamide

DMSO dimethylsulfoxide

Dppf 1,1´-bis(diphenylphosphino)ferrocene

EDG electron-donating group

ET-CORMs enzyme-triggered carbon monoxide-releasing molecules

EWG electron-withdrawing group

GC/MS gas chromatography-mass spectrometry

HRMS high resolution mass spectrometry

ISC intersystem crossing

NIR near infra-red

NMR nuclear magnetic resonance

P packing parameter

PBS phosphate buffer saline

125

List of Abbreviations

PGSE pulsed gradient spin echo

PhotoCORMs photochemically triggered carbon monoxide-releasing molecules

PSS photostationary state

Rh hydrodynamic radius

RMs reverse micelles

ROS reactive oxygen species

SANS small angle neutron scattering

SAXS small angle X-ray scattering

TEA triethylamine

THF tetrahydrofuran w water loading x water-to-surfactant molar ratio

Xanthos 4,5-bis(diphenylphosphino)-9,9-dimethylxanthene

126

Curriculum Vitae

7. Curriculum Vitae

Lenka Filipová Email: Date of Birth: 18th September 1987 [email protected]

Education 2013-present Ph.D. candidate Masaryk University, Faculty of Science, Brno Organic Chemistry, supervision by Petr Klán Thesis: Development of Photoactivatable Compounds

2011-2013 Master degree (Mgr.) Masaryk University, Faculty of Science, Brno Organic Chemistry, supervision by Petr Klán

Thesis: Synthesis and Study of Photoactivatable Amphiphiles

2007-2011 Bachalor degree (Bc.) Masaryk University, Faculty of Science, Brno Chemistry, supervision by Dominik Heger

Thesis: The Influence of Ions on the Acidity of Frozen Aqueous Solutions

Employment 2014-present Research specialist Research Centre for Toxic Compounds in the Environment (RECETOX), Faculty of Science, Masaryk University, Brno

Teaching Organic Chemistry I - seminar Chemical synthesis – laboratory

List of Publications (1) Filipová, L.; Kohagen, M.; Štacko, P.; Muchová E.; Slavíček, P.; Klán, P.: Langmuir 2017, 33 (9), 2306–2317 (2) Martínek, M.; Filipová, L.; Galeta, J.; Ludvíková, L.; Klán, P: Org. Lett. 2016, 18 (19), 4892–4895. (3) Krausková, Ľ.; Procházková, J.; Klašková, M.; Filipová L.; Chaloupková, R.; Malý, S.; Damborský J.; Heger, D.: Int. J. Pharm. 2016, 509 (1–2), 41–49.

127

Curriculum Vitae

List of Conference Contributions (1) Filipová, L.; Štacko, P.; Klán, P.: Cyanine Dyes Substituted at Heptamethine Chain Accessed by Ring-Opening of Pyridinium Salts. 53rd Advances in Organic, Bioorganic and Pharmaceutical Chemistry „Liblice 2018“, Lázně Bělohrad, November 2018 (2) Filipová, L.; Kohagen, M.; Štacko, P.; Muchová, E.; Slavíček, P.; Klán, P.: Photoswitching of Azobenzene-Based Reverse Micelles above and at Subzero Temperatures. 52rd Advances in Organic, Bioorganic and Pharmaceutical Chemistry „Liblice 2017“, Lázně Bělohrad , November 2017 (3) Filipová, L.; Kohagen, M.; Štacko, P.; Muchová, E.; Slavíček, P.; Klán, P.: Synthesis and Study of Photoresponsible Amphiphiles Containing the Azobenzene Moiety. 51rd Advances in Organic, Bioorganic and Pharmaceutical Chemistry „Liblice 2016“, Lázně Bělohrad , November 2016 (4) Filipová, L.; Štacko, P.; Klán, P.: Synthesis and Study of Photoresponsible Amphiphiles Containing the Azobenzene Moiety. Central European Conference of Photochemistry (CECP), Bad Hofgastein, Austria, February 2016 (5) Filipová, L.; Štacko, P.; Klán, P.: Synthesis and Study of Photoresponsible Amphiphiles Containing the Azobenzene Moiety. European Symposium on Organic Reactivity (ESOR), Kiel, Germany, September 2015 (6) Filipová, L.; Štacko, P.; Klán, P.: Synthesis and Study of Photoresponsible Amphiphiles Containing the Azobenzene Moiety. Central European Conference of Photochemistry (CECP), Bad Hofgastein, Austria, February 2014

128

List of Appendices

8. List of Appendices

Appendix 1A: Filipová, L.; Kohagen, M.; Štacko P.; Muchová E.; Slavíček P.; Klán P.

Langmuir 2017, 33, 2306–2317 130

Appendix 1B: Filipová, L.; Kohagen, M.; Štacko P.; Muchová E.; Slavíček P.; Klán P.

Langmuir 2017, 33, 2306–2317 – Supporting Information 143

Appendix 2A: Martínek M.; Filipová L.; Galeta J.; Ludvíková L.; Klán P. Org. Lett. 2016,

18, 4892 – 4895 168

Appendix 2B: Martínek M.; Filipová L.; Galeta J.; Ludvíková L.; Klán P. Org. Lett. 2016,

18, 4892 – 4895 – Supporting Information 173

Appendix C: NMR spectra of compounds from Chapter 2.3. and 2.4. 192

129

Appendix C

Appendix 1A

Filipová, L.; Kohagen, M.; Štacko P.; Muchová E.; Slavíček P.; Klán P. Langmuir 2017,

33,2306–2317

(https://pubs.acs.org/doi/abs/10.1021/acs.langmuir.6b04455)

192

Article

pubs.acs.org/Langmuir

Photoswitching of Azobenzene-Based Reverse Micelles above and at Subzero Temperatures As Studied by NMR and Molecular Dynamics Simulations † ‡ § † § § † ‡ Lenka Filipova,́, Miriam Kohagen, Peter Stacko,̌ Eva Muchova,́Petr Slavícek,̌ *, and Petr Klań*, , † ‡ Department of Chemistry and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic § Department of Physical Chemistry, University of Chemistry and Technology, Prague, Technická5, 16628 Prague 6, Czech Republic

*S Supporting Information

ABSTRACT: We designed and studied the structure, dynamics, and photochemistry of photoswitchable reverse micelles (RMs) composed of azobenzene-containing ammonium amphiphile 1 and water in chloroform at room and subzero temperatures by NMR spectroscopy and molecular dynamics simulations. The NMR and diffusion coefficient analyses showed that micelles containing either the E or Z configuration of 1 are stable at room temperature. Depending on the water-to-surfactant molar ratio, the size of the RMs remains unchanged or is slightly reduced because of the partial loss of water from the micellar cores upon extensive E → Z or Z → E photoisomerization of the azobenzene group in 1. Upon freezing at 253 or 233 K, E-1 RMs partially precipitate from the solution but are redissolved upon warming whereas Z-1 RMs remain fully dissolved at all temperatures. Light-induced isomerization of 1 at low temperatures does not lead to the disintegration of RMs remaining in the solution;

Downloaded via MASARYK UNIV on November 12, 2018 at 17:22:24 (UTC). however, its scope is inﬂuenced by a precipitation process. To obtain a deeper molecular view of RMs, their structure was characterized by MD simulations. It is shown that RMs allow for amphiphile isomerization without causing any immediate signiﬁcant structural changes in the micelles. See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

■ INTRODUCTION CTAB/water/chloroform-d system.7 According to this model, Reverse micelles (RMs) are self-organized assemblies of the reverse micelles are formed after the structural reorganization surfactant molecules (amphiphiles) in nonpolar solvents in of linear premicellar aggregates. Only a few studies have been which the polar heads of amphiphiles are oriented toward the performed to investigate reverse micelles at subzero temper- water cores, whereas the outer shell is composed of their atures. Several works showed that water is expelled from the micellar water core (water shedding) at subzero temper- hydrophobic chains. Many applications of reverse micelles, such 8−10 11 as microreactors for chemical and biochemical reactions,1 drug atures, including a CTAB/water/chloroform-d system. delivery vehicles,2 biocatalysis,3 the stabilizing of metastable This phenomenon can be suppressed by the fast cooling of ﬃ 11,12 proteins,4 and the synthesis of nanoparticles,5 have been reverse micelle solutions at su ciently low temperatures. presented in the literature. Amorphous ice encapsulated via the hydrogen-bonded polar The nature and behavior of reverse micelles under diﬀerent heads of AOT molecules was reported to form for relatively small conditions have been extensively investigated over the past few years. The mechanism of multistep formation of reverse micelles Received: December 13, 2016 in nonpolar solvent, known as Eicke’s association model,6 was Revised: February 14, 2017 recently experimentally validated in a micellization study of a Published: February 24, 2017

Appendix 1B

Filipová, L.; Kohagen, M.; Štacko P.; Muchová E.; Slavíček P.; Klán P. Langmuir 2017,

33,2306–2317

Supporting Information

Photoswitching of Azobenzene-Based Reverse Micelles at Above- and Subzero Temperatures Studied by NMR and Molecular Dynamics Simulations

Lenka Filipová,1,2 Miriam Kohagen,3 Peter Štacko,1 Eva Muchová,3 Petr Slavíček,3* Petr Klán1,2*

1 Department of Chemistry, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic 2 RECETOX, Faculty of Science, Masaryk University, Kamenice 5, 62500 Brno, Czech Republic 3 Department of Physical Chemistry, University of Chemistry and Technology, Technická 5, 16628 Prague 6, Czech Republic

* E-mail: [email protected], Phone: +420-54949-4856, [email protected], Phone: +420-22044-4064

Instrumentation 144 1H, 13C and DOSY NMR measurements 144 Size of reverse micelles 145 Critical micelle concentration 145 Synthesis and experimental procedures 146 MD simulations 149 NMR spectra 151 Photoisomerization kinetics of the E→Z process 156 obs Dependence of wwater, core on x for an equilibrated 1/water/chloroform-d system 156 NMR experiments with RMs 157 References 167

143

Appendix 2A

Martínek, M.; Filipová, L.; Galeta, J.; Ludvíková, L.; Klán P. Org. Lett. 2016, 18, 4892–4895.

(https://pubs.acs.org/doi/abs/10.1021/acs.orglett.6b02367)

144

Letter

pubs.acs.org/OrgLett

Photochemical Formation of Dibenzosilacyclohept-4-yne for Cu-Free Click Chemistry with Azides and 1,2,4,5-Tetrazines Marek Martínek, Lenka Filipova,́ Juraj Galeta, Lucie Ludvíkova,́ and Petr Klań* Department of Chemistry and RECETOX, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

*S Supporting Information

ABSTRACT: Photochemical generation of dibenzosilacyclohept-4-yne 3 from the corresponding cyclopropenone 1 and its copper-free click reactions are reported. Steady-state irradiation, kinetic, and transient absorption spectroscopy studies revealed that strained alkyne 3 is rapidly (<5 ns) and eﬃciently (Φ = 0.58−0.71) photoreleased from 1 and undergoes remarkably fast, selective, and high-yielding 1,3-dipolar cycloaddition with benzyl azide (∼20 M−1 s−1) or [4 + 2] inverse- electron-demand Diels−Alder reaction with 1,2,4,5-tetrazines (∼260 M−1 s−1) in both methanol and acetonitrile.

ngle-strained cycloalkynes are important molecular motifs Scheme 1. Synthesis of Cyclopropenone 1 A for click chemistry.1 Their high reactivity due to the ring strain allows for their efficient copper-free click chemistry via facile 1,3-dipolar cycloaddition2 with various substrates, for example, azides or tetrazines.1a When such reactions occur in biological systems and do not interfere with native biochemical processes, they are termed bioorthogonal reactions.1a,3 However, the rates of these cycloadditions are generally lower than those of Cu-catalyzed processes.4 To increase the rates of strain-promoted cycloadditions, fi ff commercially available tetrachlorocyclopropene in the presence structural modi cations to the cycloalkyne sca old have been ff explored, including efforts for the preparation of highly strained of a Lewis acid and subsequent in situ hydrolysis a orded cycloheptynes.5 The demand for reasonably stable but still strained cyclopropenone derivative 1 in an overall chemical reactive cycloalkynes resulted in the development of temporary yield of 30%. protection of the triple bond by its complexation with Cu(I) The absorption spectra of 1 in methanol and acetonitrile salts6 or octacarbonyldicobalt.7 possess two intense close-lying absorption bands (in methanol: λ ε ≈ × 4 −1 3 −1 Popik and co-workers, who combined rapid photorelease of max = 337 and 354 nm, 2.6 10 mol dm cm , Figure λ ε ≈ × 4 S13; in acetonitrile: max = 336 and 353 nm, 1.8 10 the triple bond of dibenzocyclooctynes from cyclopropenones −1 3 −1 via clean and efficient decarbonylation with a subsequent Cu- mol dm cm , Figure S14), analogous to those of structurally similar bicyclo[6.1.0]nonatrien-9-one derivatives.8a Compound free click reaction, demonstrated an elegant solution to spatial fl λ 8 1 is only weakly uorescent ( max = 387 nm in methanol; and temporal control of the reactivity of cycloalkynes. Bertozzi fl and co-workers studied photorelease of analogous dibenzose- Figure S13); the quantum yield of uorescence was below 0.01. 9 We found that 1 in both solutions undergoes partial but still lenacycloheptynes and their in situ trapping with benzyl azide. fi Here we present the click chemistry of a highly strained signi cant (<20%) photochemical degradation during the seven-membered dibenzosilacyclohept-4-yne derivative, photo- acquisition of the spectra; therefore, only three scans of freshly prepared samples were averaged to obtain the final spectrum. generated from the corresponding cyclopropenone, with benzyl ffi azide and 1,2,4,5-tetrazines. The mechanism and kinetics of the Upon irradiation at 375 nm, e cient photodecarbonylation of 1 was observed in both methanol (Φ = 0.58 ± 0.01) and reaction steps were evaluated by steady-state and transient Φ ± absorption spectroscopy experiments. acetonitrile ( = 0.71 0.01) to give silacycloheptyne 3 (Scheme 2). This compound exhibits two distinct bands (in The 4-silabicyclo[5.1.0]octatrienone derivative 1 was pre- λ λ pared using a procedure analogous to that employed for the methanol: max = 315 and 334 nm; in acetonitrile: max = 316 and 335 nm) in its absorption spectra (Figure S15), analogous synthesis of a 4-selenabicyclo[5.1.0]octatrienone derivative by 8a Bertozzi and co-workers9 (Scheme 1). The first step involved a to those of cyclooctyne derivatives. coupling of two 3-bromoanisole molecules using n-butyllithium and dichlorodimethylsilane to give dimethyldiphenylsilane 2. Received: August 8, 2016 Two consecutive Friedel−Crafts alkylation reactions of 2 with Published: September 14, 2016

Appendix 2B

Martínek, M.; Filipová, L.; Galeta, J.; Ludvíková, L.; Klán P. Org. Lett. 2016, 18, 4892–4895.

SUPPORTING INFORMATION

Photochemical Formation of Dibenzosilacyclohept-4-yne for Cu-Free Click Chemistry with Azides and 1,2,4,5-Tetrazines

Marek Martínek, Lenka Filipová, Juraj Galeta, Lucie Ludvíková, Petr Klán*

Department of Chemistry and RECETOX, Masaryk University, Kamenice 5, 625 00 Brno, Czech Republic

Table of Contents

Materials and methods 174 Synthesis 175 Determination of quantum yields and rate constant 178 NMR spectra 179 Absorption spectroscopy studies 185 Irradiation experiment with 1 186 Light sources 187 Kinetic studies 188 References 191

173

Appendix C

Appendix C: NMR Spectra of Compounds from Chapter 2.3. and 2.4.

Confidental content

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