Asterios, Nitika Elisabeth Marc

• B Khurana

P Gierlich

A Meindl

L C Gomes-Da-Silva

M O Senge

Crystals of photoactive BODIPY dye in pattern spreading as tree branches in a flask Other publications

he met me in India as a part of visit to my university and a very brief interaction with him, ended up being the best thing ever happened to my life. He has been a guiding force for me to secure and complete my PhD, your care, and your vision for your students to succeed is why I have grown to my career and goals. Thanks for believing in me and trusting me to be part of his amazing tetrapyrrole group also known famously as the "Senge group" .

Nothing in life is to be feared, it is only to be understood. Now is the time to understand more, so that we may fear less. -Marie Skłodowska-Curie I seem to have been only like a boy playing on the seashore and diverting myself in now and then finding a smoother pebble or a prettier shell than ordinary, whilst the great ocean of truth lay all undiscovered before me. -Isaac Newton It is strange that only extraordinary men make the discoveries, which later appear so easy and simple. -Georg C. Lichtenberg iii

DEDICATION

To the loving memory of my younger brother Vishav who passed away 9 years ago…years ago when I found your unfinished bucket list, that gave me a dream to live for and a goal to achieve. I hope you were here, and I miss you a lot….

To my parents, Seema and Yogesh who dreamt my dreams, always being a guiding force for my lows and a laughing soul for the best I got. If it weren't for you, I couldn't have made it so far. Thanks for all the unconditional support and believing in me and being patient with the times we have been apart.

To my loving elder brother Dr. Sumit, you have been my inspiration for perseverance and dedication to get what you aim for. Thanks for being an ear to my endless rants.

To my friends, the new and the old ones, who always cheered for me; thanks for being there through thick and thin: Dhruv, Anant, Neha, Manish, Aditi, Shweta, Karolis, Jessica, Susan, Elisabeth, Marc, Asterios, Nitika, Alina, Chris, Aurore, Piotr, Claire, Emma, João, Harry, Dáire, Manu, Zoi, Nidia, Jérémy, Veronica, Abdechakour. Thanks for sharing all those good moments with me and making life something special

ACKNOWLEDGEMENTS

Since my childhood the only thing that remained constant to my curiosity was the practical science which prompted me to pursue it as a career but there was always a barrier to my dreams, first lack of support to local PhD programs in science in my country and secondly as an international student to support myself and to manage things staying so far away from home.

With Professor Senge's guidance I was able to apply for a great opportunity of a PhD position in project POLYTHEA whilst doing a M.Sc. research project with him. The road to PhD was still a few baby steps away when after being on the wait list, I finally got this prestigious PhD position in project POLYTHEA with Professor Vincent Sol in University of Limoges and Professor Dr. Mathias O. Senge in Trinity College Dublin.

Over the past three years of my PhD (2019-2022) several treasured memories will always stay with me, engraved in my heart. All the while handling the COVID repercussions and restrictions, the possibility of my manuscript work coming together is not only me but involves many different people, who have contributed in various but imperative ways. This journey would not have been possible without the support of all the lovely people including my mentors, my colleagues, and my family.

Firstly, I express a special gratitude to all the members of the jury and especially, Dr. Francesca Giuntini and Pr. Santi Nonell for accepting to be the referees and for investing time in the evaluation of my work.

I would also like to acknowledge the coordinator and manager of the project, Dr. Stéphanie Leroy-Lhez for giving me a chance to showcase my abilities and to learn and develop my scientific skills in Europe. A big thanks to Ms. Aurore Berthier for being a helping hand in literally everything, my experience in France has been amazing and easy because of you.

With a deep sense of gratitude, I would like to convey thanks to Pr. Vincent Sol, our UNILIM lab-head, for accepting me to join his group as a part of this project. The first thing he told me was I was the first ever Indian PhD student in chemistry with his lab group and with that I wanted him to feel proud to have me onboard. It has been my pleasure to be a member in your team. My French experience of PhD has been a smooth and enjoyable one with your support and guidance throughout. I will always fall short of words to express my gratitude towards Pr.

Senge, who is my secondary supervisor, without him nothing was possible. A few years back 1. Introduction

The "ANTIBIOTIC" era

Sir Alexander Fleming was a pioneer in the field of antibiotics; with the discovery of penicillin in 1928, he set forward a new era in medicine, allowing effective treatments for a range of microbial infections. Since then, as shown in Figure 1.1, several families of antibiotics have been approved by the European Union (EU) and United States (US) regulatory bodies. These medicines act by targeting the cell wall and disrupting synthetic protein processes in the microbiome. [1] Figure 1.1. New classes of antibiotics. Colors refer to the principal target; red: inhibiting folic acid synthesis; blue: inhibiting the cell wall synthesis; pink: inhibiting the protein synthesis; green: inhibiting the DNA or RNA synthesis [1] In the modern world, antibiotics are essential to handle infections by microbes and their related pathology. [2] However, antibiotics have become the "panacea" in medicine, used pre-emptively to treat infections of non-bacterial nature in humans, and to promote growth rates in animals. [3] Microbial infections that have been responsible for several deaths throughout centuries, threaten to return to a state of lacking effective treatment.

As microorganisms replicate at rapid rates and mutate, an accelerated evolution provides antimicrobial drug resistance; with selective pressure this becomes increasingly prevalent throughout the microbe population. [4] Resistance to drug treatment can co-occur via the formation of biofilms that penetrate the extracellular polymeric substances (EPS) auto-produced by the produced by the microorganisms, hindering penetration of the relevant antimicrobial drug. [5] As such, the adaptability of microorganisms to eradication strategies presents a danger to human health in addition to the environmental problems caused by stray biota. [6] Therefore, the American academy of periodontology (AAP) has recently embarked on a best evidence consensus (BEC) model of scientific inquiry that every new antimicrobial treatment is a valuable weapon to combat these infections caused by planktonic and biofilm-embedded microorganisms. [7] The Gram stain allows a broad categorization of bacteria into two categories based on their surface composition. An example of such clinical drug-resistant isolates are the bacterial species of Gram-positive methicillin resistant-Staphylococcus aureus (MRSA), cephalosporin-resistant Gram-negative Escherichia coli, or carbapenem-resistant Gram-negative Pseudomonas aeruginosa, each a concerning source of global pandemics and environmental issues. [8,9] Acinetobacter baumannii, a Gram-negative bacterium often associated with hospital-acquired infections, acquires multi-drug resistance, which causes problems by triggering obstinate infections in wounds and burns, notably for people affected during military conflicts. [10] Other Gram-negative bacteria such as Aggregatibacter sp., Porphyromonas sp. and Fusobacterium sp. and Gram-positive ones, such as Staphylococcus sp. and Streptococcus sp., lead to inflammation of tooth-supporting tissue, a disease known as periodontitis, and are often associated with antibiotic resistance. [11] Relative risks are localized; these have been mapped on a linear scale from lower values (green) to higher values (red), as shown in Figure 1.2. [12] Figure 1.2. Global distribution of the relative risk of an emerging infectious disease event caused by drug-resistant pathogens. Categorized by standard deviations from the mean and mapped on a linear scale from green (lower values) to red (higher values). [12]* *Reproduced with permission

Drug-resistant infections have a significant, continuing impact on health globally, uncountable mortality and morbidity and causing productivity loss of at least 1.5 billion euros in the EU and of about 5 billion dollars in the US per annum. [13] The most prominent example is the current world situation involving a fight against COVID-19 and the SARS-CoV-2 virus, which has been evolving since its first report and has attained mutant forms. [14] The most recent as reported is the Omicron variant, which has caused a devastating loss of human lives and economic certainty. [15] Traditional treatments for viral infections involving chloroquine and its analogues (hydroxychloroquine) or remdesivir drug-based treatment have been used to treat COVID-19 patients. [16] In response to the deaths related to COVID-19 disease, effective new antiviral drugs or vaccinations have been expedited, but no strategy provides a 100% effective approach to maximize protection against these pathogens. [17] An increased risk to human health is additionally caused by fungal strains (e.g., Candida albicans) that have developed drug resistance towards common antifungal agents such as amphotericin B, ketoconazole, itraconazol and fluconazole. [18] Even the protozoan parasitic infections undergo incomplete or ineffective treatment of leishmaniasis with drugs as prescribed, enhancing the drug resistance to as evaluated for L. mexicana and L. braziliensis isolates. [19] Therefore, it is of utmost importance and priority to find alternatives to traditional antimicrobial therapies. In April 2014, the World Health Organization (WHO) had issued a warning on being on the cusp of a "post-antibiotic era" [20] . The growing resistance to many antibiotics in recent years and the emergence of multidrug-resistant bacteria has diverted attention towards alternative antibacterial therapies. Among the novel, alternative therapies are bacteriophages, naturally occurring or synthetic antimicrobial peptides, and in recent years antimicrobial photodynamic therapy (aPDT) has been employed. [21,22] 1.1.1.

A brief history of antimicrobial photodynamic therapy (aPDT)

In ancient times people have employed a traditional use of light combined with a photoactivated entity to treat skin diseases. It has been documented that in ∼1200-2000 BC, Egyptian and Chinese physicians and Indian ayurvedic practitioners have used a combination of plant-based extracts and sunlight to treat skin infections, although many of these treatments are not scientifically validated. [23] The modern approach to antimicrobial photodynamic therapy (aPDT) was first reported in 1900 by Oskar Raab in Munich when he observed that Paramecium caudatum was killed after staining with PS, acridine orange and subsequent exposure to light of suitable wavelengths. [24] Additional experimentation emphasized the importance of oxygen in the photodynamic action against the facultative anaerobic species Proteus vulgaris. [25] Figure 1.3 shows the results of the first antimicrobial photodynamic experiment using the eosin dye as a PS in combination with sunlight to kill Streptococcus bacteria. [26] In 1903, H. von Tappeiner and A. Jesionek treated skin tumors with topically applied eosin and white light. This phenomenon was coined "photodynamic action". [26,27] At the onset of the 20 th -century, Danish physician Niels Finsen proved the usage of photodynamic therapy by applying heat and light filtered through a carbon lamp to treat cutaneous Lupus vulgaris. [28] Antimicrobial applications of the photodynamic process have been reignited in response to the first drug-resistant infections in the healthcare sector during the early 1990s. [29] 1.1.2.

Antimicrobial photodynamic therapy (aPDT)

Photodynamic therapy (PDT) is clinically used as a minimally invasive therapeutic tool;

cytotoxicity is induced by a light source that potentially exerts selective activity against malignant cells and infected tissue. This approach has found potential anti-microbial uses in recent times against all types of microorganisms, such as Gram-positive and Gram-negative bacteria, fungi, parasites and even viruses. [30,31] APDT alternatively known as photodynamic inactivation (PDI), or lethal photosensitization, or photodynamic antimicrobial chemotherapy (PACT), provides a possible way to eliminate microbe species efficiently, cost-effectively, and more importantly, without the associated mechanism of resistance. [32] aPDT has several advantages over antibiotics. Firstly, aPDT offers precise targeting, and is considered triply site-specific due to: (1) preponderant association/uptake of PSs by the target cells compared to non-target cells, (2) the pharmacodynamic inertia of nonirradiated PSs, as well as (3) the site-confined irradiation of the infected area. [33] Consequently, systemic toxicity is largely absent outside the irradiated, PS-replete zone.

Secondly, compared with antibiotics, no resistance is developed against aPDT PSs; even repeated treatment with aPDT did not lead to the selection of resistant strains. [34] This is due to the drug-light interval (the time between the PS and PDT) being too short for bacteria to develop resistance, and the near-absent dark toxicity, resulting in bacteria not having to engage adaptive survival mechanisms against the PSs. It is also difficult for bacteria to 'sense' that the oxidative stress emanates from the otherwise non-toxic PS, so any metabolic adaptations are directed elsewhere (e.g., antioxidant defense machinery).

Thirdly, targeted cells are eviscerated after PDT, rendering them too damaged to confer cross-generation adaptivity. Lastly, aPDT does not target a single site in bacteria, much different from conventional antibiotics. The reactive oxygen species (ROS) generated by aPDT targets various bacterial cell structures and other metabolic pathways. [35] In short, the treatment vector is perfect: precise, hyper-effective and unable to be guarded against, evaded, or learned from, while benign to surrounding tissue. These reasons underlie the potential utility of aPDT in combatting resistant strains in a non-minimally invasive and patient-friendly manner.

aPDT against bacteria

The potential of aPDT against diseases of bacterial origin is vast, engaging many of the advantages as a treatment demonstrated above. This potential has not been exploited due to (a) the discovery and development of advanced antibiotics; (b) non-effectiveness against some well-known pathogens, especially Gram-negative bacteria, which have poor results with known, traditional photosensitizing agents many of which are structurally like the crystal violet Gram stain. [36] The aPDT efficiency is therefore dependent on the structural entities of the cell wall of the target bacteria as described in detail in this section.

Gram-positive and Gram-negative bacteria

The bacterial domain has been classified into two subgroups based on their difference in the cell wall structure and the chemical composition, namely, Gram-positive, and

Gram-negative bacteria, as differences in their Gram stain staining. [37] As shown in Figure 1.3, the bacteria cell membrane is a complex multi-layered protective structure providing an innate immunity against the unpredictable and hostile environment. These structural differences between the cell walls of both kinds of bacteria reflect differences in their biochemical composition. [38] Figure 1.3. Schematic representation of the cell wall and cytoplasmic membrane structure in Grampositive (left) and Gram-negative (right). [38]*

*Reproduced with permission

For Gram-positive bacteria, the principal constituent of the cell wall is multiple protective layers of peptidoglycan. N-Acetylglucosamine and N-acetylmuramic acid held together by peptide bridges constitutes the amino sugar backbone of such strains. This makes up 40% to 80% of the cell wall weight, depending on the species, and has high tensile strength. Teichoic and teichuronic acids are also part of the cell wall of Gram-positive bacteria. Teichoic acids are polyol phosphate polymers, such as polyglycerol phosphate and polyribitol phosphate. Both acids are linked covalently to peptidoglycan. [38] The peptidoglycan layer is also present in the cell wall of Gram-negative bacteria. Still, in contrast to Gram-positive bacteria encased with multiple layers of peptidoglycan, Gram-negative ones have only a single to a few macromolecular sheets. Indeed, the peptidoglycan makes up about 5% by weight of the Gram-negative cell wall, but it still plays a vital role as a solid protective barrier outside the cell membrane. The outer membrane component of Gram-negative bacteria has the predominant presence of lipids and proteins and some polysaccharides extending into the aqueous environment.

This complex lipid-polysaccharide layer is known as lipopolysaccharide (LPS), a high molecular weight, powerfully negatively charged molecule. The outer membrane of Gram-negative bacteria has lipoprotein covalently linked to the diamino acid of the peptidoglycan, forming a complex called peptidoglycan-lipoprotein. Therefore, the lipoprotein is the bridge that joins the outer membrane to the cell wall peptidoglycan layer. Transport proteins (porins), another critical component of the cell wall of these bacteria, allow the passage of molecules through the outer membrane. This cell wall feature forms water-filled channels that work as a general or substrate-selective conduit for free diffusion of specific hydrophilic molecules, such as sugars and amino acids, and inorganic ions. The negative charge on LPS molecules provides a polyanionic external surface partly neutralized by divalent cations, such as Mg 2+ and Ca 2+ and is a connecting bridge adjacent to LPS molecules. The periplasmic space between the cell wall and the cell membrane is also important because it is a zone of tremendous enzymatic activity inside. [39] 1.1.4.

Mechanism of action in aPDT

The three essential elements of aPDT are: 1) a photosensitizer, 2) a source of light, and

3) molecular oxygen. [40] Figure 1.4. Schematic illustration of the main photophysical and photochemical processes involved in PDT. [117] The photophysical processes taking place during aPDT are illustrated by the energy level diagram in Figure 1.4. The PS used in aPDT is a ground electronic singlet state (S0) and, after irradiation with light of a suitable wavelength, is excited to a short-lived singlet state (Sm). The photosensitizer can return to the ground S0-state, emitting the absorbed energy as fluorescence (used for diagnosis and imaging) or undergo intersystem crossing to the excited triplet state (T1). [41] This transition is generally spinforbidden, indicating that suitable PSs must have a high triplet-state quantum yield. The T1-state is sufficiently long-lived to take part in different chemical reactions but will return to the S0-state via phosphorescence or a non-radiative decay dependent on lifetime.

Thus, our photosensitizer acts as a photocatalyst, promoted from its ground state to electronically excited state returns to that energy level via these three main pathways.

Depending on the reactive species formed, aPDT is classified into two types. Type I relies on the interaction between the excited triplet state of photosensitizer ( 3 PS*) and substrates from the target tissue. New radicals generated from photosensitizers and substrates interact with molecular oxygen and other molecules in the environment. In type II, the direct reaction of 3 PS* and molecular oxygen gives rise to singlet oxygen [ 1 O2], a highly reactive form of oxygen. [42,43] 1.1.

aPDT active photosensitizers

As established in the previous section a photosensitizer is a chromophore-based compound that can absorb photons from the incident light, thus producing ROS, a highly active species. For clinical aPDT use, an ideal photosensitizer would possess the following properties:

• chemical purity and ease of synthesis for commercial use;

• photostability, high phototoxicity, low dark toxicity;

• chemical stability for transportation, storage and reconstitution;

• rapid exclusion from target tissues to minimize any side effect, minimal photosensitivity towards the subcutaneous tissues, good pharmacokinetic activity;

• the high quantum yield for ROS ( 1 O2) generation;

• preferential association with bacteria versus mammalian host cells;

• suitable pharmacokinetics, and accumulation in bacteria or binding to the bacterial cell envelope. [44,45] Typically, PSs are excited within the red part of the visible range <650 nm of light or closer to the near infra-red. First-generation PSs such as Photofrin are hampered by minimal light penetration of tissue. Second generation PSs, which absorb at longer wavelengths (>630 nm) like some synthetic or natural PSs where light penetrates tissue deeper without being impeded by other endogenous biomolecules, have a better activity for aPDT. Alternatively, third-generation photosensitizers, targeted PSs such as Visudyne (clinically used) are under development with enhanced sensitivity towards longer wavelengths of the illuminating light and better target specificity. [46] PS binding to the bacterial cell and uptake is dependent on the type of bacterial species.

Due to the unique structure of the cell envelope, Gram-positive pathogens are much more susceptible to anionic and neutral PS because of the thick but porous peptidoglycan layer on the outer surface. Gram-negative bacteria are less prone to take up exogenous compounds due to the extra outer cell membrane and the permeability barrier imparted by lipopolysaccharides. [47] Efficient antimicrobial candidate when tested in combination using the time-kill approach is achieved at a minimum of 2-log decrease or more in the number of colony-forming units. [48] PSs used in aPDT are classified into four groups based on their structure and origin: synthetic dyes such as phenothiazinium dyes and borondipyrromethenes difluoroboradiazaindacene (BODIPY) dyes, tetra-pyrrole designs such as porphyrins, phthalocyanines and chlorins, natural PSs such as curcumin, hypericin, flavin, chlorophyllin and phenalenone derivatives and nanostructures based on fullerenes and titanium oxides. Table 1.1 illustrates commonly studied antimicrobial PSs and sampled photodynamic properties.

Synthetic dyes

Phenothiazinium is a subgroup of synthetic dyes, and these PSs are planar, tricyclic, and aromatic molecules. which have been used in medicine for over 100 years. [70] The commonly used phenothiazinium dyes are methylene blue (MB) (1.1) and toluidine blue (TB) (1.2) as shown in Figure 1.6, have been used for their efficacy in aPDT due to the presence of the cationic charge, they have an affinity to bind to both Gram-negative and Gram-positive bacteria. MB and TB have been studied extensively for their effectiveness on planktonic bacteria and were tested against biofilm structures. [71] Functionalization on phenothiazinium such as methylation induces a greater photoinactivation and an increased production of ROS species. Recently, new derivatives such as dimethyl methylene blue (derived from MB) due to the presence of a high cationic charge have shown to be effective against bacterial cells. [72] Other synthetic dyes are eosin Y (1.3), erythrosine (ERY) (1.4) and Rose Bengal (RB)

(1.5) as shown in Figure 1.5; these have also been used as PSs for aPDT. These are anionic charged xanthene dyes derived from fluorescein. [73] These dyes show an absorption band in the green wavelength range of 480-550 nm and have shown significant activity against the production of biofilm growth as well and though they have demonstrated lower activity against Gram-negative bacteria, they are effective against the growth of Gram-positive bacteria. Compound 1.1 has been used in the sterilization of blood products since it has a high chemical affinity to nucleic acids and thus can be used against virus, including HIV, hepatitis. 74] Compound 1.2 has been predominantly used in the treatment of oral infections, for sterilizing dental cavities and root canals, and for treating periodontitis. [75] Borondipyrromethenes difluoroboradiazaindacene (BODIPYs) (1.6) dyes as shown in Figure 1.6 have shown potential for antimicrobial inactivation and are used as components of antimicrobial coatings. [76] BODIPY dyes can be chemically modified to introduce negative or positive charges on the scaffold of this PS, and exhibit high singlet oxygen quantum yields, acting predominantly according to the Type II mechanism. [77] BODIPYs are well established as fluorescence imaging dyes in diagnostics as they possess intense color and fluorescence, high extinction coefficients, and do not undergo photobleaching. [78] Chemical modification of BODIPY dye leads to an increased excited triplet state and enhanced ROS formation. With appropriate modifications, BODIPY dyes have been shown to have increased strength of interaction with bacterial target structures. [79] Figure 1.6. Chemical structure of BODIPY (1.6) dyes.

Tetrapyrrole structures

Porphyrins (1.7) are a group of naturally occurring, intensely colored, tetrapyrrolic compounds as shown in Figure 1.7, which have found applications in several fields, ranging from fluorescence imaging to medicine. [80] Porphyrins have a strong absorption band around 400 nm (Soret band), and further absorption peaks between ~500-700 nm (Q-bands). Many porphyrin derivatives with different side groups have used as effective PSs for aPDT. Introduction of cationic charge increases the predilection of the PS to the bacterial cell wall, hence mediating a better aPDT action against the bacterial strains. Porphyrins show several advantages over rival PSs; the absorption of light at 400 nm allows significant energy to be delivered, and the ease of modification around its molecular periphery allows the synthesis of charged derivatives with enhanced action against the microbes. An exemplar cationic PS based on porphyrin is TMPyP•4(I -) (5,10,15,20-tetrakis(1-methyl-4-pyridyl)porphyrin tetraiodide) (1.8) as shown in Figure 1.8, possessing a 4+ charge across the peripheral pyridinium rings. [81] It has been demonstrated that this species has shown to be more active than the neutral/uncharged adduct of same porphyrin as against B. cereus endospores formation. [82] Figure in Figure 1.9. [82] This functionalization further enhances the action against bacterial endospore growth. This chemistry highlights opportunities to make more efficient PSs based on porphyrin macrocycles, using rational design, especially of ionic charge, to enhance antibacterial action with functionalities introduced in the macrocycle. Phthalocyanines (1.11) consist of four pyrrolic rings, connected via nitrogen atoms as shown in Figure 1.10. These PSs have higher absorption in the far-red region of the spectrum (650-700 nm) and hence has been used for applications where deep-tissue penetration by light is required. [83,84] Figure 1.10. Chemical structure of phthalocyanine (1.11).

The addition of central metal ions, like Zn 2+ (zinc phthalocyanine) (ZnPc) or Al 3+

(aluminium phthalocyanine) (AlPc) have shown an enhancement in the production of singlet oxygen; ZnPc demonstrates a reported affinity for Gram-positive bacteria. [85,86] ZnPc, in conjunction with cationic groups or anti-membrane agents such as polymyxin or EDTA (ethylenediamine-tetraacetic acid), has been demonstrated as effective against Gram-negative bacteria. [87] Like the porphyrin macrocycle, phthalocyanine is amenable to chemical modification at the core and periphery, and enhancement of the aPDT activity of this macrocycle is actively researched. A few notable examples of rationally designed molecules have been synthesized, which includes the water-soluble octa-cationic zinc Pc (1.12) shown in Figure 1.11, presumed to be effective against both Gram-negative and Gram-positive bacteria. [88] Also, it has been shown that cationic

ZnPcs have a potency to eliminate E. coli from blood products, making it advantageous in sterilization. [89] Figure 1.11. Chemical structure of octa-cationic phthalocyanine ligand (1.12).

Chlorins are second generation photosensitizers, having high photodynamic efficiency. E.g., chlorin e6 (1.13) (Ce6, Figure 1.12) -mediated aPDT has been shown to completely suppress the growth of Porphyromonas gingivalis, Fusobacterium nucleatum, and Capnocytophaga gingivalis and has also been shown to inhibit the human dental plaque species. [90] A significant reduction of clinical signs of redness and bleeding on probing (BOP) in periodontitis-induced beagle dogs has also been shown via the PS, Ce6. Radachlorin® is an aqueous solution of three photosensitizers, including chlorin e6 (90-95%) as the major ingredient, with sodium chlorin p6 (5-7%);

this has shown an effective PDT action against Streptococcus mutans. [START_REF] Fekrazad | Photodiagn[END_REF] Advantageous photophysical characteristics of this water-soluble chlorin mixture include the high quantum yield of singlet oxygen (75%), low toxicity in the dark, intense absorption band in the middle-red part of the spectrum, quicker kinetics in comparison with Ce6 alone, and high phototoxicity make it uniquely powerful as a PDT therapeutic agent. [92] N

Natural/semi-synthetic PSs

There are many naturally occurring compounds extracted from plants and other organisms which have potency as a photosensitizer upon absorption of white light or UV-A. Hypericin (1.14), curcumin (1.15) and chlorophyllin (semi-synthetic) (1.16) as

shown in Figure 1.13 are three naturally derived compounds that have been extensively studied as photosensitizers over the years. [93,94] Hypericum perforatum or St John's-wort is a plant that also demonstrates antiviral, antidepressant, antibacterial and antitumor characteristics. [95] It has been shown that Streptococci mutants, Lactobacilli mutants, and Propionibacterium acnes. [96,89] . The photoactivity of hypericin against clinically isolated Gram-positive MRSA and E. coli produces extended-spectrum ß-lactamases. [89] Curcumin (1.15) (Figure 1.13) is a natural PS isolated from the root of a plant called Curcuma longa having absorption in the range of 405-435 nm. It possesses a series of biological and pharmacological functions, being an antioxidant, having anti-inflammatory and anti-microbial properties and assisting in wound healing. [97] These innate properties of curcumin make it a favorable PS for the treatment of periodontal diseases. In terms of photo-biocidal effects, studies indicate that curcumin is 300 times more effective against the Gram-positive S. aureus than the Gram-negative E. coli and Salmonella typhimurium. [98] Chlorophyllin (CHL) (1.16) refers to a family of derivatives of chlorophyll; cyclic tetrapyrroles with the heteroaromatic character of the underlying chlorin system. The reactivity of the peripheral substituents of this macrocycle allows facile modification of this molecule to introduce targeting functionalities. [99] CHL itself is not ideal as a PS, since the paramagnetic Cu 2+ hinders the formation of long-lived excited states. [100] PSs derived from Cu-free CHL as shown in Figure 1.14, functionalized with three tertiary amines or three quaternary ammonium groups possessing the amphiphilic character with positive charge favors the compound to bind to the cell membranes and walls and aid in intracellular localization in mitochondria. [94] Figure 1.14. Synthetic route to chlorin with tertiary or quaternary ammonium groups. [94] Popularity of this PS derives from the ease of semi-synthetic preparation; hydrophilic CHL can be isolated from water insoluble chlorophyll. Further synthetic steps allow these compounds to be tuned to optimum properties as a better PS.

Limitations of aPDT

It has been established that the advantages offered by aPDT could contribute to resolving the troubling issue of antimicrobial resistance and mutations by eliminating a spectrum of infectious microbes. However, the adoption of aPDT-active PSs still encounters significant challenges; these compounds are often poorly soluble in aqueous media, may lack photostability, and differ significantly in their pharmacological properties. [101] As planar, aromatic compounds the PSs are prone to π-π stacking and hydrophobic interactions, and majority of them aggregate in aqueous solution. [102] The localization of PSs on the microorganism surface is dependent on several factors, with charge, molecular size, concentration of PSs, lipophilicity, and cell wall composition each playing a role. [103] Light-induced destruction of pathogens has proven more effective for Gram-positive bacteria due to their "single-layered" cell wall/membrane structure allowing deeper penetration of the PS. [104] The presence of a well-organized and thick outer membrane in Gram-negative bacteria renders these more resistant towards aPDT; the thickness of the outer membrane also limits the penetration of PS through the cell membrane and wall. This presents the necessity to design targetspecific photosensitizers against such pathogens.

A multitude of pathogens can be targeted by aPDT; however, this technique is nonselective with respect to intracellular molecular targets -proteins, lipids, and nucleic acids are each affected by induced oxidative stress. [105] For cationic PSs, due to electrostatic mismatch between human cells and PSs, poor affinity of the sensitizer and limited efficacy of the treatment has been reported. [106] This can be seen as a positive; aPDT can target and eliminate pathogenic microbes while minimizing damage to human cells. Chlorophyll based PSs, even though having excellent aPDT potential, can often kill non-target organisms posing a risk to the ecosystem when used for environmental applications. [107] Photosensitivity, a common side effect of application of PSs to humans, confers side-effects such as redness and swelling. Rare allergic reactions have similarly been observed. [108] Thus, a PS must be formulated in a way to minimize aggregation. [109] In addition, it should ideally be target-specific, biodegradable, cost-effective, non-toxic in the absence of light, and deliver PSs in their monomeric form to the target tissue. [110] Research has shown that selective targeting of the PSs to the target cell or tissue could be done by designing target specific delivery platforms based on peptides (polymyxin). [111] , Introduction

The advantage of incorporating a PS into the drug delivery system (DDS) is improved bioavailability, solubility, permeability, and selectivity. These DDS provide protection of PS against external factors including light and temperature. [116] The following properties allow the incorporation of a large amount of hydrophilic and hydrophobic PSs for better target selectivity. Further, these formulations could be functionalized structurally via the chemical attachment of ligands, enzymes, and antibodies to combat the very issue of microbial resistance. [117] The physicochemical characteristics of DDS affect the interaction with the biofilm matrix, as the smaller the size of the nanocarrier the better is the penetration into the biofilm. The surface charge of such a system is an important factor for the drug's efficacy, as the cationic charged DDS have greater penetration in the biofilm matrix as compared to anionic or unloaded platforms. [118] The size, loading capacity, functionalization of the DDS, as well as inherited properties of the PSs, modulates targeting in the cell membrane of the bacteria as well as the biofilms and can increase the antimicrobial activity under aPDT conditions. [119] 1.3.2.

Types of drug delivery system

Drug delivery systems are based on encapsulation of the drug in a biocompatible matrix, either a solvent, polymer, gel or lipid. Selected examples of drug delivery systems which have previously been used for aPDT are listed in Table 1.2 and expanded upon in this section. [120,137,146,150,153,160,174]

Hydrogels

Hydrogels are a group of hydrophilic polymeric materials reticulated via chemical bonds and interactions, named for their ability to hold a large quantity of water within their threedimensional (3D) network structures. [120] Today's understanding of polymeric hydrogel materials dates back to studies with poly(vinyl alcohol) (PVA) cross-linked with formaldehyde in the late 1940s, used for biocompatible implants for humans and marketed as "Ivalon". [121] In 1958, Danno synthesized a polyvinyl alcohol polymer using gamma-radiation, forming a cross-linked network of a hydrogel. [122] A key advance occurred in 1960, when Wichterle and Lim reported a polymeric cross-linked system of macromolecules based on poly(2-hydroxyethyl methacrylate) (pHEMA). [123,124] This material is currently used to manufacture soft contact lenses for eyes, the first successful mass-marketed cross-linked hydrogel material, now with decades of use demonstrating safety for humans. In the 1950s and 1960s Katchalsky worked extensively on polymeric networks and established the possibility to transfer chemical energy into mechanical strength. [125] This inspired in the early 1970s the development of stimuli specific secondgeneration hydrogel materials. These materials are characterized by stimuli-responsive activity with variation of temperature, pH, or the concentration of molecules in solution, affecting the cross-linking properties and the pore size of the hydrogels, allowing an additional targeting vector for drug-delivery. Examples of temperature-sensitive polymeric networks include poly(isopropylacrylamide) (PNIPAAm), poly(N-(2hydroxypropyl)methacrylamide) (PHPMAm) and polyethene glycol (PEG)-polyester block copolymers; advances in the mid-1990s exploited physical interactions to crosslink the polymers to form hydrogel networks, enhancing the mechanical strength as well as degradation and thermal stability of the polymeric systems. [126] A third generation of hydrogels were established as an example of stereo-complexed materials by blockcopolymers via forming an inclusion complex possessing metal-ligand coordination in addition to peptide cross-link interactions. Hydrogels with incorporated metals are widely used for sensing applications utilizing photoluminescent hydrogels for pH sensing. [127,128] Continuing research into the dynamics of these types of DDS has led to a proliferation of these stimuli responsive ''smart polymers''; polymeric cross-linked hydrophilic networks possessing tunable properties which can be triggered externally, or by physiological responses. [129,130] Common techniques include in situ cross-linking of the hydrogels, radical polymerization, formation of double-network polymers, by a combination of natural and synthetic polymeric materials which allow one component to be selectively modified, or by forming composite polymeric formulations with small inorganic molecules. [131] These materials possess ideal platform properties for biological applications, chiefly controlled drug delivery (PS) to the target site.

The polymer source may be natural or synthetic, but they function in the same way, are used to carry PSs loaded from the dispersion medium (i.e. water) to the target. [132] Macro-scale gels (>1 mm) persist at the site of infection for a longer interval of time due to their inherent low fluidity whereas microgels and nanogels, due to their small size, enable the PSs to effectively reach the target sites while retaining the advantages of a gel hence are emerging as an effective carrier material in aPDT. [133,134] Gels can be further modified with different functionalities or with other targeting agents altering the biological or physical properties, allowing improved biodistribution, pharmacokinetics, cell uptake, and target selectivity. [134] Antimicrobial hydrogels have been used widely in clinical settings; well-known examples include bactericidal silver-hydrogel coatings for medical device sterilization and their application in wound dressings to support wound healing. [135] Both hydrophobic and hydrophilic PSs can be included in a microgel/nanogel system via physical (encapsulation) or chemical (conjugation) methods as shown in Figure 1.15.

Due to their fluidity, hydrogels can deliver more active material to the site of infection than a decorated surface. The size of the nanogel mesh, the flexibility of the polymeric chains, the reticulation density, and their ability to incorporate and bind with water molecules modulates the structural and mechanical properties of hydrogels and can be expected to similarly affect medicinal properties. [136] Incorporation into these polymeric hydrogel networks have helped to overcome the issue of solubility of common photosensitizers in the aqueous medium, reducing aggregation of the PSs and reducing the dark toxicity as induced by some PSs. [120] Cross-linked polyvinyl alcohol (PVA)-borate hydrogels have been used as an effective aPDT DDS. When conjugated with two cationic charged PSs -MB and TMPyP they allowed effective treatments for wounds infected with both planktonic cells and biofilmgrown MRSA. [137] The antibacterial activity of MB incorporated into polyacrylamide (PAA) based hydrogel matrices showed an effective activity against bacterial suspensions and biofilms. [138] Photodithazine (PDZ) was formulated in a natrosol-based hydrogel to improve its topical application against the fungal species of Candida albicans, Candida glabrata, and Candida tropicalis biofilms. Gram-negative bacterium P. aeruginosa. [141] Surface localization of TMPyP electrostatically bound in acrylate hydrogels has been shown to decrease bacterial colonization and could possibly be used as intraocular lens biomaterials to prevent endophthalmitis (ocular infections). [142] Linear polymeric amidoamines have been used to prepare conjugates with other aPDT active PSs such as the BODIPY-NMe dye and have been shown to be active against the Gram-negative E. coli Gram-positive S aureus, respectively. [143] Iontophoretic release of PSs from a polyelectrolyte hydrogel for potential wound healing loaded on poly(methyl vinyl etherco-maleic acid) gel with either MB or TMP was achieved and it showed an effective aPDT potency against MRSA and Burkholderia cepacian. [144] Figure 1.17. Schematic drawing of the photodynamic inactivation of microorganisms by singlet oxygen generated by the photosensitizer immobilized on a polyacrylamide support. [140] 1.

Bio-polymer derived hydrogels with PSs

Compared with synthetic polymers, biopolymers have received greater attention, due to their biocompatibility, bioactivity, and biodegradability. [145] The chemical structure of some biopolymers used to synthesize hydrogels -those incorporating PSs active for aPDT -are illustrated in

Cellulose

Cellulose (1.23) is the most abundant biopolymer in nature, occurring principally in the cell walls of plants. It is a high molecular weight polymer with the empirical formula [C6H10O5]n which is renewable, non-toxic, colorless, and odorless. [146] Structurally, cellulose is composed of D-glucopyranose units linked via β(1-4) linkages. This polymer is hydrophilic, biocompatible, biodegradable and is mechanically robust, all advantageous for DDS use. These properties also account for its use in a vast array of fields and, being a carbohydrate, it possesses high compatibility with biological systems. [147] A chemically modified derivative of cellulose acetate has been used to encapsulate PSs such as RB and TB as a biopolymeric coating to investigate the ability against microbes in hospital conditions. [148] A white fluorescent lamp (with two prominent bands of λ = 545 and 610 nm) was used to eradicate disease-causing bacteria E. coli, bacteriophage Clostridium difficile, the yeast Candida albicans, and MRSA. These photoactive coatings were prepared by dissolving cellulose acetate polymer in acetone, and both PSs (RB and TB) were added in appropriate concentrations. The tests against the microbial populations showed PSs can retain their antimicrobial properties when embedded in the biopolymer since the levels of cell death achieved (up to five-millionto-one (6.7 log) reduction) should be more than sufficient for surface disinfection in a hospital setting.

Cellulose impregnated with the porphyrinic PS, 5,10,15,20-tetrakis(4-N,N,Ntrimethylammoniumphenyl)porphyrin (TMAP 4+ ) or its zinc(II) complex (ZnTMAP 4+ )has similarly shown photo-bactericidal effects. [149] glucosamine. Interaction of the polycationic charges of this platform with the microbial surface potentiates the aPDT of PSs. This polymer is biocompatible, biodegradable, and displays an attractive broad-range antimicrobial activity and is easily modifiable due to a large number of hydroxyl and free amino groups. [152] It has been shown that ERYgrafted CS is efficient against the bacteria and fungi such as P. aeruginosa (Gramnegative), S. mutans (Gram-positive), and Candida albicans (fungi). [153] Furthermore, the chitosan-coated indocyanine green (ICG)-loaded poly(lactic-co-glycolic acid)

(PLGA) showed adherence to the bacterial cell wall of Gram-negative P. gingivalis, as well as other periodontal pathogens, Fusobacterium nucleatum and Aggregatibacter actinomycetemcomitans. [154] It is thus proving to be an efficient tool against the faecalis and P. aeruginosa. [155,156] It was shown that this formulation adheres to bacteria cells and to biofilm extracellular polymeric substances (EPSs) which results in a higher uptake into the biofilms, enhancing the aPDT potency.

Chitosan formulation in combination with MB is efficient against planktonic bacterial growth and biofilm formation of Gram-negative bacteria P. acnes. [157] The antimicrobial action of this formulation was achieved at a concentration of 12.5 µg/mL of MB and it was observed that reduction of 1.9 log10 biofilm was obtained at a concentration of 75 µg/mL MB. It was seen that the formulation exhibits adhesion to the skin patches thus making this formulation a very effective platform for localized infections.

Decoration of chitosan with zinc phthalocyanine-colistin (1.30) results in an antibacterial formulation for both treatment and sterilization, particularly against multidrug-resistant Gram-negative strains such as P. aeruginosa. [158] This kind of formulation, as shown in

Hyaluronic Acid

Hyaluronic acid (HA) (1.27), as shown in Figure 1.19, is a naturally occurring, biocompatible anionic biopolymer having unique physicochemical and distinctive biological activities. [159] It is a linear polysaccharide skeleton of β-1,4-linked D-glucuronic acid (β-1,3) N-acetyl-D-glucosamine disaccharide and is found as a non-sulfonated glycosaminoglycan (GAG) in the extracellular matrix (ECM) of higher animals. [160] Unmodified HA has found an essential utility in applications involving drug delivery, tissue engineering, viscous-supplementation and it has natural wound healing properties. [161] Chemical modification of HA can help tailor the physicochemical properties and in vivo residence time of HA while retaining its natural biocompatibility and biodegradability. [162,163] Functional modifications of carboxylic groups on HA was used to formulate polymer-drug conjugates via a covalent conjugation with the synthesized PS with HA and itis a powerful platform to improve aPDT. [164] Such platforms can be used as an alternative to traditional antimicrobial medicines to treat localized infections.

Formulation obtained by crosslinking collagen-hyaluronic acid with 1-ethyl-(3-3dimethylaminopropyl) carbodiimide hydrochloride (EDC.HCl), fabricated with the antibiotics tobramycin or ciprofloxacin assist wound healing and sterilization. [165] A formulation fabricated with 0.4 mg/ml of ciprofloxacin has exhibited cytotoxic effects on fetal human dermal fibroblasts, making these polymeric platforms ideal candidates to be explored for aPDT.

Curcumin, as explained earlier, is a naturally occurring antibiotic molecule which has been explored for its aPDT potential; solid dispersions of curcumin with methyl-β-CD, HA and hydroxypropyl methylcellulose (HPMC) or an intercalated system with both HA and HPMC have been formulated. [166] The intercalated formulation has been tested for in vitro photoinactivation of E. faecalis and E. coli, displaying a log 6 reduction of colony for both strains at a concentration of about 0.5-10mM, under illumination with blue light. This formulation is therefore promising for wound treatment and sterilization.

Silver nanoparticles (AgNPs) are known to exhibit an antibacterial effect, but the major disadvantage of such NPs is the underlying toxicity to human cells. [167] Antibacterial applications of such NPs could be enhanced by binding these molecules onto HA, making a strong synergistic framework of photosensitive metal-organic hybrids. [168] This platform has shown an enhanced biocompatibility due to presence of negatively charged HA preventing the bleaching of the Ag ions. The target bacteria secrete an enzyme (hyaluronidase) degrading the HA, triggering the release of the positively charged nanoparticles of silver and enhancing the antimicrobial activity of this platform preserving the ability to generate reactive oxygen species (ROS). This platform has shown an enhanced effect against multidrug-resistant bacteria infected wounds.

HA based composite hydrogel conjugated with an antimicrobial-peptide [AMP, KK(SLKL)3KK] with HA via a Schiff's base reaction was synthesized to attain a longlasting antimicrobial activity. [169] The primary amines are derived from peptides (lysine residues) and aldehyde groups from HA. This hydrogel formulation has shown an effective injectability, high biostability, and an impressive mechanical strength. These composite systems have a shown an excellent broad-spectrum antibacterial activity in vitro and in vivo.

Lipid Delivery Systems

It is important to have high production of ROS species to achieve an improved and effective aPDT. The hydrophobic nature of the PSs as synthesized often causes aggregation in aqueous media. This aggregation reduces the aPDT efficacy by depleting the formation of singlet-oxygen species due to a self-quenching effect in the excited state. [170] Lipid delivery systems (LIP), encapsulating and protecting non-target cells from the PS cytotoxicity, can preserve the monomeric forms of the PSs and result in improved aPDT. [171] These kinds of DDSs have a number of advantages; an enhanced loading capacity of PSs compared to other systems, allowance for the incorporation of both hydrophilic and hydrophobic PS species, better biodegradability, biocompatibility and the synergistic activities of positively charged and highly fluid components of LDS, all of which enhance uptake and phototoxicity. [172,173] 1.

Liposomes

Liposomes (specifically phospholipids) as shown in Figure 1.21, are derivatives of LIPs, spherical vesicles capable of forming a closed bilayer structure. These can be varied on the order of micrometers and have been widely investigated due to their lipidic structure's similarity with human cell membranes. [174,175] Liposomes are used as a pharmaceutical tool to improve the efficacy of aPDT efficacy and drug targeting due to their non-toxic nature, biocompatibility with human cells and ability to incorporate nearquantitative amounts of PSs, and promoting controlled release. [176]

*Reproduced with permission

The flexibility inherent in the structural chemistry of such vehicles allows the incorporation of several hydrophilic and hydrophobic PSs, making them ideal DDS in a clinical setting. Physical properties such as the composition of lipids, bilayer fluidity, and a surface charge may influence the drug behavior and modulate their pharmacokinetic profile and biodistribution into the body. [177] It has been shown that liposomes behave differently in biological systems depending on their surface charge (positive, neutral, or negative) and their ability to bind to the surface. [178] Cationic liposomes have been used to incorporate aluminium chloride phthalocyanine, this aPDT formulation used against cariogenic bacteria in dental cavity disinfection. [179] Against Porphyromonas gingivalis, using a fluorescent lipid incorporated in the liposome membrane, antibacterial effect of aPDT mediated by free or liposome-encapsulated zinc phthalocyanine was also evaluated. [180] The PS used showed a decreased dark toxicity as individual PS, enhanced photochemical stability, and a strong absorption in the red region in this matrix, which may facilitate light penetration into tissues. Almost 99% of liposomes bonded to P. gingivalis, while the neutral liposomal vehicles showed poor binding. Thus, in accordance with the results, cationic liposomes are expected to retain the photosensitizer when attached to the biofilm, prolonging the period of attachment and acting as a local reservoir of the PS.

Objective and outline of the manuscript

Even though aPDT is emerging as a potential tool against the worlds tormenting issue of increasing number of drug-resistant pathogens, most PSs used for conventional therapies lack selectivity, target specificity and can undergo self-quenching in biological media due to aggregation. Several strategies to enhance the activity of such PSs have been discussed above which allows for a better solubility and selectivity of the PSs towards the target microbe. This can include combining the PS with a drug delivery system or chemically altering the molecular skeleton of the PS with functionalities which induces better potency towards the site of infection.

Overall, our aim in this manuscript is to explore the multiverse of aPDT potential and enhancement of PSs via different functionalization strategies which includes conjugating with carrier molecules or post functionalization of these molecular probes itself. Finally getting a standout strategy for a better aPDT. To achieve this, In Chapter 2 we synthesized two potential PSs which have been reported in literature for their antimicrobial activity but lack target selectivity and are relatively less soluble or aggregate in aqueous media. With an aim to work on these drawbacks these PSs were conjugated with a drug delivery platform. Hyaluronic acid biopolymer offers an excellent advantage of being biocompatible and has been used in skin industry for formulations of products. This polymer has anionic molecular skeleton with functionalities that could be modified to introduce our PSs covalently onto them. These PSs have a primary amine functionality and are covalently conjugated onto the COOH functionality of hyaluronic acid biopolymer as shown in Figure 1.22. Lastly in Chapter 4 with an aim to build up a library of fluorescent aPDT active PSs and addressing the innate solubility issues, low selectivity with the bacterial culture, different functionalization strategies were applied to obtain derivatives of PSs. Our major focus for this part of the manuscript is to recognize the efficiency of such scaffolds for aPDT and to give a standout synthetic strategy for future exploration of similar probes in aPDT.

Thus, we report a synthesis of library of BODIPY dyes which have been modified on their molecular skeleton to address the PS related issues for an efficient activity in biological evaluations including inhibiting the growth of the infective bacteria and their biofilms. The protocol involved synthesizing different meso-N-heterocyclic BODIPY dye scaffolds and further introducing cationic charge or synthesizing zwitter ionic species onto the N-heteroatom using different functionalities which make them ideal candidates to interact with a wider stratum of microbes as shown in Figure 1.23. The protocol involves successive introduction of cationic charges and water solubilization groups (methyl, carboxylate and etherate) onto the BODIPY. This strategy allows converting the hydrophobic BODIPYs to the corresponding water-soluble dyes without hurting their optical properties. These derivatives were tested for production of singlet oxygen species using the DPBF degradation assay to access the photobiological activity of such compounds. Finally, these derivatives were subjected to MIC/MBC analytical assays to evaluate the aPDT potential of these derivatives with Gram-strains of bacteria along with the inhibition and eradication of biofilms protecting the infection causing bacteria. Another strategy with aim to make water soluble derivatives with multiple carboxylate functionalities on the BODIPY scaffold B-C derivatives at the boron atom were synthesized under Grignard reaction condition to yield derivatives as shown in Figure 1.24. These derivatives were further subjected to synthesis protocol of N- All the studies and synthesis done accomplishes the goal of our manuscript to develop new strategies to make PSs more efficient enhance their activity for aPDT. As has been established, porphyrin and chlorin based photosensitizers (PSs) undergo photoactivation with a suitable wavelength of light, inducing an oxidative damage to the bacterial cell wall via production of reactive oxygen species (singlet oxygen and free radicals). [1,2] 2.1.1.

B N N F F Ar B N N F F Ar Ar = N N N N N N S N N N N N X X

Cationic porphyrins

An efficient PDT against microbes (aPDT) requires that the PS is co-located with the target cells prior to irradiation with light. Cationic PSs have been the most popular candidates used for aPDT, due to an enhanced and effective interaction to the charged surface of the bacterial cell wall, especially gram-negative species which have proved difficult to eliminate. [3] In the literature, there are several examples of efficient aPDT when bacteria are treated with cationic porphyrins. TMPyP, a typical cationic porphyrin, has been studied intensively for its antimicrobial activity; research has indicated that it can also induce photodynamic inactivation (PDI) of E. coli via production of singlet oxygen under illumination with light. [4] TMPyP has also shown very promising activity against radiation resistant Dinococcus radiodurans bacteria under illumination with blue light. [5] A newly synthesized porphyrinbased photosensitizer, Exeporfinium chloride (XF-73) (2.1) as shown in Figure 2.1, has demonstrated an efficiency as a PS against the MRSA species and exploring the potential of such PSs for sterilization per se. [6] aeruginosa and E. coli. [7] These compounds are shown in Figure 2.2. Typhoid is a common and recurring infection in humans, caused by contact with untreated water bearing the parasite. It has been established that photoinactivation of Salmonella typhimurium can be achieved by the cationic ruthenium derivative of 5,10,15,20-tetra(4pyridyl)porphyrin (RuTPyP) (2.4) or its Zn(II) complex (ZnRuTPyP) (2.5) complexes, as shown in Figure 2.3. [8] These derivatives display effective formation of reactive oxygen species (ROS) under irradiation of light with substantial singlet oxygen quantum yields of 0.02 and 0.36 for RuTPyP and ZnRuTPyP, respectively in DMSO measured under irradiation with 200 mW cm -2 halogen lamp . These examples establish that cationic porphyrins are promising candidates to be tested and explored for their aPDT potential. Introduction of further modifications to the porphyrin periphery allows to explore the structure/activity relationships beyond the introduction of charge.

N N H N H N R R MeO R R = N N Br Br

Chlorins

Energy is the critical impetus for life; most of the energy available to lifeforms flows from the sun to the earth as visible light. As such, the pigments which perform this function are both readily available and evolutionarily optimized; none is better known than the central photoactive molecule of the plant photosystem, chlorophyll-a. [9] This molecule is so central to life, that its color is synonymous with life itself ("greenery"), its expression acting as the epicenter of supporting and balancing all ecosystems on land.

Chlorophyll-derived chlorins are heterocyclic aromatic moieties related to porphyrin, having three pyrrole rings and one reduced 3,4-dihydropyrrole (pyrroline) ring coupled via methine linkages. This molecule, especially the 'special pair' thereof in photosystem-II,

N NH N HN N Ru N Cl N N N N Ru N Cl N N N N Ru N Cl N N N N Ru N Cl N N N N N N N N Ru N Cl N N N N Ru N Cl N N N N Ru N Cl N N N N Ru N Cl N N N Zn 2.4 2.5 . P F F F F F F . P F F F F F F
has become important to photochemistry, as its absorption maxima lies at the red end of the visible region (660-800 nm) where human tissue is generally translucent, which indeed makes these moieties an ideal PS to establish a higher depth of treatment in PDT. [10] Several chlorin PS candidates have been derived from this natural-source, or synthesized from simple chemical units, and have been evaluated for their therapeutic action as PDT agents. Semisynthetic chlorins can be classified on the basis of their chemical constitution into two different classes: hydrophobic PSs, based on pheophorbide (Pba) (2.6a) and its derivatives and amphiphilic PSs, chlorin-e6 and chlorin p6 (Cp6) (2.6b) and hydrophilic purpurin-18 (Pp-18) (2.6c) derivatives. [11] The chlorin Pp-18 has been used for PDT; with an advantageous absorption peak at 695 nm; this PS allows deeper penetration at the site of infection. [12] This molecule, however, possesses a chemical instability due to the presence of an anhydride ring which readily reacts with nucleophiles. Upon reaction with water, this yields another photosensitizer, chlorin p6, as shown in Scheme 2.1. gave chloro-substituted or bromo-substituted derivatives with regioselectivity, optimizing the photophysical activity (i.e. PLQY) of this PS through the heavy atom effect. [14] Figure 2.4. Numbered molecular structure of purpurin 18 (Pp-18) and positions of frequent modifications. The Pp-18 molecule is usually modified to improve its photochemical properties, for example, by halogenation (pink and blue dots) or nitration (a blue dot), or derivatized by conjugation reactions with peptides or other tumor cell-targeting molecules (an orange dot) and cyclic anhydride to form imides (a green dot). [13,14] Several functionalization of Pp-18 makes this PS more usable for PDT, with targeting groups and stability adjustments; the most stable derivative Cp6 has been used for PDT intensively. Cp6 was first introduced as a potential anionic PS in the 1980s and its chemical skeleton has three acidic carboxyl groups in the lower periphery of the molecule and has a absorption maxima at around 400 nm and is known to be efficient for production of singlet oxygen species (quantum yield ~0.60 in ethanol). [11,15] This class of Pp-18 derivative PS molecules has great potential applications in aPDT that have been explored only to a limited degree in literature studies.

It has been demonstrated that the related chlorin derivative Ce6 showed complete suppression of infections caused by microbes Porphyromonas gingivalis, Fusobacterium nucleatum, and Capnocytophaga gingivalis. [16] This derivative has also shown a 90% efficacy to eliminate the human dental plaque species. [17] The commercially available and clinically proven radachlorin is composed of Ce6, Cp6, and purpurin-5; each has a high quantum yield of singlet oxygen, low dark toxicity, absorption maxima in visible spectrum and high phototoxicity, making this combination a unique pharmacological agent. As a photosensitizer for aPDT this preparation has been shown to be active against Streptococcus mutans and for treatment of periodontitis. [18]

Formulations PSs for aPDT

The PSs described above have innate properties of being PDT-active against the infectious and world-troubling microbes, with charges on the molecular skeleton enhancing the interaction with bio-membranes and aiding solubility in biological media.

A conjugated formulation with a nano-scale drug-carrier can potentially enhance the selectivity of the PS in both action and solubility, augmenting these high-potency molecules interactions with the infected microbial surfaces and ensuring rapid elimination from the site of infection. [19,20]

Cationic PSs-formulations for aPDT

With enhanced solubility, no dark toxicity, and sufficient adherence time for the interaction of the photosensitizer at the site of infection, aPDT with this binary PS/DDS approach is gaining clinical interest. [21] In recent years, the research and development of antimicrobial drug-delivery systems has been extensive, and the inclusion of PSs into these platforms is a promising approach for aPDT. [22,23] This combination is ideal for wound dressings, where a higher level of local drug concentration on surface, and minimal diffusion, is ideal. [24] An example of a cationic porphyrin conjugate prepared by the Sol group at the University of Limoges, with 5-(4-aminophenyl)-10,15,20-tris(4-N-methylpyridinium)porphyrin triiodide with polypeptide polymyxin is shown in Scheme 2.2. This binary system possesses positively charged residues on a PDT agent, in addition to a known Gramnegative targeting antibiotic motif, allowing simple formulation with two methods of causing cell death. [25] Scheme 2.2. Synthetic route of cationic porphyrin-Polymyxin B conjugate.

The conjugate showed an effective inhibition of Gram-positive strain of Staphylococcus aureus and most important against an enhanced inhibition against the Gram-negative P. aeruginosa and E. coli. It was demonstrated that the porphyrin-peptide conjugate adhered to the cell walls of the Gram-negative strains selectively, and caused damage, induced by production of singlet oxygen.

Another carrier platform for tricationic porphyrins was prepared on cellulose paper using a 1,3,5-triazine derivative as a linker and it was evaluated for its antimicrobial potential as shown in Scheme 2.3. [26] Antimicrobial activity of this conjugate was evaluated against Gram-positive S. aureus and Gram-negative E. coli. This formulation successfully inhibited the microbes under light illumination, making this carrier a novel platform with cationic PSs for aPDT.

N NH N HN N N N NH O N O O H N O O OH O O N + HCTU, DIEA N H O SH H N O N H NH 2 H N O N H O H 2 N HN O O NH O OH O HN H N O NH NH 2 O NH 2 O OH Polymyxin-B N H O S H N O N H NH 2 H N O N H O H 2 N HN O O NH O OH O HN H N O NH NH 2 O NH 2 O OH N O O H N O Cationic poprhyrin-polymyxin conjugate .3I - Scheme 2.
3. Synthetic route to photo-antimicrobial filter paper. [26]

Chlorin-formulations for PDT

As explained earlier Pp-18 can readily undergo ring opening of the anhydride ring to form a chlorin p6 form of the PS, and this property has been explored for reaction with a nucleophilic agent forming a conjugate with poly-L-lysine (pl-cp6). It allowed inactivation of the methicillin-resistant Staphylococcus aureus (MRSA) and P.

aeruginosa. Similar derivatives have been shown to inhibit the hyperinflammatory response of infections; hence, mediating wound healing process, reducing risk of infection complications. [27] The polycationic conjugates of Ce6 with polyethylene (PEI) (PEI-ce6) showed an inhibition of pathogenic S. aureus, E. faecalis (Gram-positive) and E. coli (Gramnegative) bacteria and the yeast Candida albicans after irradiation with appropriate spectrum of light. [28,29] These conjugates have also shown an inhibition of multi-drug resistant localised Acinetobacter baumannii bacterium. Purpurin 18 (Pp-18) when reacted with polyethyleneimine (PEI) has shown to yield a water-soluble conjugate of chlorin shown in Scheme 2.4. [30] Scheme 2.4. Synthesis of photosensitizers bearing PEI 600 Da (a), 2000 Da (b), 25,000 Da (c). [30] This formulation of the PS with PEI has been covalently grafted on cellulose nanocrystals (CNCs) obtained by hydrolyzation with sulfuric acid solution (64% w/w) as shown in Scheme 2.5.

Scheme 2.5. Synthesis of photosensitizer-CNCs derivatives. [30] * These CNCs grafted with Pp18-PEI have been evaluated with the keratinocyte cell line (HaCat) using MTT assay and the complex showed an optimal PDT activity.

Hyaluronic acid polymer conjugate with PSs

HA (2.16) biopolymer is a non-sulfated glycosaminoglycan composed of repeating units of the disaccharide β-1,4-D-glucuronic acid-β-1,3 N-acetyl-D-glucosamine. [31] It possesses an inherent biocompatibility and bio-functionality and has beneficial physicochemical properties, such as high-water retention and viscoelastic properties, making this biopolymer a right choice for bio-applications in several fields of medicine specifically in cosmetology. [32] The HA chains can be cross-linked, either chemically or physically, to form DDS. The physicochemical properties, stability, and half-life of the native HA can be improved by modifying its structure and forming a conjugate. [33] After the reaction, the HA conjugates can maintain the biocompatibility and biodegradability that characterize the unmodified material. [34] The disaccharide units of HA possess three sites that may undergo chemical modification: the carboxyl group, hydroxyl group, and N-acetyl group as shown Figure 2.5. [35] HA conjugated with chlorin derivative chlorin e6 (Ce6) has been evaluated for its efficiency in PDT. [36] The formulation with HA was prepared with reducible disulfide bonds (HSC) of the HA biopolymer encapsulating perfluoro hexane (PFH) within the PFH@HSC nanoparticles as illustrated in figure 2.6. These conjugates act via passive and active tumor-targeting. Nanoparticle photoactivation from "OFF" to "ON" is mediated by redox responsiveness of the conjugate. With an advantage of acting as a diagnostic

O O NH O OH OH O O HO HO n OH 2.16
dye, the chlorin is incorporated into PFH@HSC; this conjugate acts as a dual imagingguided PDT in treatment of hypoxic tumors.

*Reproduced with permission

Several additional HA conjugates with PSs efficient for aPDT are described in chapter 1. Formulations with this biopolymer make the conjugate more biocompatible and with rapid clearance from the site of infection imploring the further study of conjugates with PSs as described above to evaluate their efficiency in aPDT. Functional modification of carboxylic groups on HA has been used to formulate polymer-drug conjugates via a covalent conjugation with the synthesized PS with HA and it is a powerful platform to improve aPDT. [33] Such platforms can be used as an alternative to traditional antimicrobial medicines to treat localized infections.

Synthesis of PSs and PS-HA conjugates for aPDT

Chlorin p6 (purpurin-18 derived) and porphyrin-based (cationic 5-(4-aminophenyl)-10,15,20-tris(4-N-methyl pyridinium)porphyrin triiodide) PSs have been synthesized in this chapter. These PSs have been established for having efficient therapeutics for aPDT against the two strains of bacteria. These PSs have been selected for synthesis and to form a covalent conjugate as they possess a primary amine function which has been used to covalently graft these PSs on the COOH functionality of hyaluronic acid (HA) platform via an amide link.

Extraction and synthesis of purpurin-18 (2.17) chlorin p6-PEI (2.18) conjugate

To synthesize the chlorin p6-PEI (2.18) conjugate we extracted and chemically modified the chlorin to obtain purpurin-18 (Pp-18) (2.17) using a previously described procedure from the blue-green algae Spirulina maxima as shown in scheme 2.6. [30,37] The resulting compound was characterized via spectroscopy which giving the characteristic 1 H spectrum of a chlorin macrocycle and the presence of an absorption band at 413 nm confirming the formation of the adduct.

Scheme 2.6. Extraction of purpurin-18 (2.17) from Spirulina maxima and chemical modification to chlorin-p6 (2.18). [30] The macrocycle of 2.17 possesses an anhydride exocyclic ring which can react very quickly with nucleophilic groups such as a primary amine (PEI). Thus, 2.17 was reacted done in propionic acid as shown in Scheme 2.7. [25] After purification by silica column chromatography, a purple powder in a literature verified yield of 4% was obtained. The formation of the PS was verified by NMR and UVvis spectroscopy with an absorbance maximum (λmaxH2O) at 415 nm. Compound 2.22 was further modified to prepare the cationic derivative via methylation with excess iodomethane (2.23), followed by reduction with H2O/TFA. This yielded the final cationic, 5-(4-aminophenyl)-10,15,20-tris(4-N-methyl pyridinium)porphyrin triiodide 2.24. Methylation and acidic hydrolysis of the acetamide group resulted in a good yield of 82%, which is in accordance with the literature. [25] These macrocyclic PSs were obtained with primary amine function, which is further conjugated with biopolymeric platform of hyaluronic acid. This resulted in the formation of covalent conjugates of HA-PS (2.25 and 2.26) which were further characterized as detailed in the following.

Differential scanning calorimetry (DSC)

The thermal analysis profiles of the dried samples (with or without PSs) were obtained as the temperature was increased from room temperature to 250 °C at a rate of 10 °C/min under N2 atmosphere. Calorimetric analysis of these conjugates yields classic thermograms (Figure 2.8.) in which we observe an endothermic peak resulting from loss in water from the conjugate and an exotherm was observed at 80 °C and at 240 °C, a characteristic feature of HA biopolymer. [38] a) b)

Scanning electron microscopy (SEM) characterization

Representative SEM images in Figure 2.9 of the dried samples (with PSs) show homogenous dispersion of the samples and cross-linking pattern with an average size of 105 ± 103 nm for 2.25 and 215 ± 105 nm for 2.26. These compounds present macroporous structures with irregular pores are an indication of the conjugated PSs with HA which is important for biomedical applications. [39] a) b)

Infra-red (IR) spectral evaluation

The covalent grafting of HA and photosensitizers that bear a primary amine function (2.18 and 2.24) was confirmed by FTIR measurements. The corresponding FTIR spectra of HA-photosensitizer-conjugates are displayed in Figure 2.10. Compared to the spectrum of HA alone (black line) which possessed characteristic absorption band around 3360 cm -1 assigned to stretching vibration of hydroxyl groups of hyaluronic acid and peaks at 2930 and 1340 cm -1 (stretching and deformation vibrations of C-H bond), the conjugate 2. 26 showed new signals that correspond to photosensitizers (1570, 1250, 810 cm -1 ). The absorption band for this conjugate at 3325 cm -1 and 1642 cm -1 can be attributed to the amide functional group that forms between the amine and carboxylic groups. The IR spectrum of the dried conjugate samples for 2.25 (Figure 2.10, red line) showed the presence of PEI characteristic peaks at 2947 and 2930 cm -1 and chlorin characteristic bands at 1574 cm -1 , 1596 cm -1 and 1644 cm -1 , respectively. [40,41]

Dynamic light scattering (DLS) analyses of conjugates

The DLS showed a 245 nm hydrodynamic diameter. After formulation of the hyaluronic acid with PSs, the hydrodynamic diameter of conjugate 2.25 increased to 342 ± 52.8 nm and for 2.26 increased to 435.1 ± 35.1 nm. The zeta potential for 2.25 was found to be -34 and for 2.26 was determined as -29.3 due to acid moieties of the hyaluronic acid. [42]

UV-vis analysis to identify the percentage conjugation of PS on HA

The UV/visible absorption properties of photosensitizers 2.18 and 2.24 remained intact after binding to HA as shown in Figure 2.11. The number of photosensitizers bound to HA was estimated by UV-visible absorption at the maximum of Soret bands (between 40 and 440 nm) using a calibration curve of free PSs 2.18 and 2.24. The relative molar percentage of photosensitizers in the conjugate was determined based on monomer concentration to be 18% for conjugate 2.26 and 3% for 2.25. The difference in yield could be explained by the low grafting reactivity of the PS, 2.18 with the condition used. Indeed, at the end of the reaction, we observed many PS in the reaction medium. The low grafting molar % of 2.18 grafted on HA is the reason for no singlet oxygen production as evaluated by ergosterol degradation assay and, hence, it was not used further for biological assays against bacteria.

Rheological behavior of the PS-HA conjugate.

As shown in Figure 2.12, non-conjugated HA, and modified HA (i.e., chlorin p6-PEI-HA As these conjugates are water soluble, these conjugates can set a landmark to explore more modifications in the PS or HA chemical skeleton to improvise the aPDT potential of such formulations.

These formulations have been evaluated for their efficacy against the microbes using the tradition growth assays and determination of inhibition concentration to identify the efficacy of such conjugate formation strategy.

Antimicrobial evaluation of HA-PS conjugates

A small bacterium can change the dynamics of the world for years, as the situation has been recently. It is important to have some potential solutions to the very basis of the infections caused by microbes. As explained earlier in this chapter a positive charge on the PS molecule enhances the binding and penetration of the PS efficiently into the cell.

With conjugated platforms as synthesized above the target specificity and biocompatibility of the formulation and overall efficiency for aPDT is expected to be enhanced.

We evaluated our conjugates for inhibition of the Gram-positive S. aureus and Gramnegative E. coli species via diffusion agar assays and MIC evaluations, to determine the efficiency of our platforms.

Diffusion test on agar

Growth inhibition studies of the formulation of chlorin p6-HA (2.24) showed a negligible The inhibition activity as against the bacteria for the conjugates is not what was expected this could be due to presence of negative charges on the biopolymer of hyaluronic acid which could neutralizes the charge on the cationic porphyrin (2.24). This could cause a reduction in the interaction of the conjugate with the bacterial cell wall.

Conclusion and future perspective

The conjugate platform cross-linked with photosensitizers has ideal properties of increased solubility in aqueous media and an ability to produce singlet oxygen (in case of CP-HA (2.26)). The conjugates obtained from synthesis and covalent linkages on HA when tested for photo bactericidal applications shows some antimicrobial activity in the presence of light. However, the presence of strong negative charges due to the carboxylic acid functions of the biopolymer decreases the interaction between the conjugate and the bacterial walls. Thus, after illumination, the singlet oxygen produced is not close enough to the bacterial walls to have a significant PDT effect. Nevertheless, these conjugates facilitate the formulation and medical application of PSs, and it could be envisaged to increase the number of incorporated PSs and/or to associate other polymers to obtain composites capable of interacting more easily with the bacterial walls. Another strategy to possibly enhance the antimicrobial action would be to use a reticulation agent like DTPA anhydride as recently published by Sol group in Limoges. [43] This strategy introduces the spacer molecule between the biopolymeric chains and use the free carboxylic groups of the HA to covalently graft the cationic PSs or chlorins as described previously or introduce the PSs via ionic interactions between the PS and carboxylate functionalities of HA and reticulating agents.

Chapter 3. Relative singlet oxygen yield of photosensitizers

Relative singlet oxygen yields via DPBF degradation assay

Background and molecular design

The basic principle of aPDT -the selective uptake of the active PS and subsequent irradiation and eradication, requires a light source of an appropriate wavelength matching the spectral wavelength of photosensitizing dye. [1] A photon of light is absorbed by a photosensitizer molecule, exciting this molecule from a singlet ground singlet state (S0) to an excited short-lived singlet state (S1). This state will undergo nonradiative transitions including an intersystem crossing (ISC), changing the multiplicity of the photosensitizer electronic state from a singlet to a longer-lived triplet state. [2] Under aPDT conditions, this PS can decay back to the ground state via two different processes as illustrated in Figure 3.1 -either by spontaneous phosphorescence or through energy transfer with proximal oxygen species. This process can produce reactive oxygen species, either oxygen-centered radicals and radical ions (type I), or its formation of an excited singlet oxygen diatom (type II). Both of these processes will occur in any given setup, and the ratio between the type I and type II processes depends on the kind of photosensitizer irradiated. [3,4] Therefore, it becomes important to quantify the generation of singlet oxygen of the photosensitizer to understand the basic parameters of aPDT efficacy of a PS.

Several singlet oxygen detection probes have been investigated and have been described in the literature.

Singlet oxygen ( 1 O2) detection probes

The detection of singlet oxygen, as distinct from the ground state diatom, requires measurement of the rate of some chemical reaction which is specific to this species.

The probe consists of a chromophore and 1 O2 receptor, in which formation of endoperoxides (EP) or 1,2-dioxetanes species disrupts the absorbing system. The most common probes used, as shown in Figure 3.2, incorporate both motifs and interact directly with 1 O2 oxygen. [5] The change in the electronic structure of the sensor molecule can be monitored using different spectroscopic techniques such as NMR, EPR, UV-vis absorption, or fluorescence (Table 3.1). [6] Figure 3.1. Reaction types after excitation of the photosensitizer to produce ROS species. [2] Table 3.1. Spectroscopic detection methods for singlet oxygen with different sensor molecules. 1,3-Diphenylisobenzofuran (DPBF), parent of the 1,3-diarylisobenzofuran class, has a highly-reactive diene core that can scavenge unstable and short-lived dienophiles in a Diels-Alder reaction. [7,8] DPBF is one of the most widely used compounds for spectrophotometric detection of singlet oxygen due to its rapid and irreversible reaction with 1 O2 providing a clear absorption and fluorescence signal. DPBF, when incorporated in model biological membranes and excited at 410 nm, shows a fluorescence emission spectrum with a maximum at 455 nm. [9] DPBF effectively suppresses the triplet state and therefore has effectively no selfphotosensitization (i.e., does not act as PS to generate 1 O2). In the presence of singlet A decrease in absorption at this wavelength is linear in proportion to the amount of singlet oxygen adduct formed. [11] The primary advantage of using the DPBF degradation assay is that it reacts with 1 O2 in an essentially diffusion-limited reaction and thus has a very high sensitivity, trapping up to 50% of all 1 O2 in alcohol/water or micellar solution. This setup is ideal for environments where only small amounts of 1 O2 are produced. [12] 3.1.2. DPBF degradation probes for PSs (relative singlet oxygen quantum yield)

Property

Our group (the Senge group) at Trinity College Dublin have published various articles in which the DPBF singlet oxygen probe method was used for determining the 1 O2 production of PSs. [13] For example, the library of BODIPY-anthracene and -pyrene dyads in Figure 3.3 showed promise for applications in photodynamic therapy; these were evaluated with the DPBF degradation probes to quantify the PSs efficiency for formation of singlet oxygen species. These dyes were functionalized to produce water-soluble derivatives to enhance the application of such dyes in PDT and interaction with biological media as shown in Figure 3.4. [14] With introduction of water-soluble groups on such dyes the production of singlet oxygen as evaluated with DPBF degradation assays remains almost unaffected; hence, removing the drawback of aggregation and quenching these derivatives become a better candidate for PDT. The singlet oxygen production is determined by measuring the relative singlet oxygen quantum yield (Ф∆) with known PSs, in this case RB. These experiments showed that all the PS-conjugated PNIPAM hydrogels produce singlet oxygen efficiently, and that the detection system functions under these conditions. The hydrogel formulation presumably reduces the aggregation of porphyrins increasing the efficiency of singlet oxygen production in PNIPAM-based hydrogels as compared to the PSs alone.

O O O O O O + 1 O 2 -"O" Endoperoxide derivative 1, 2-dibenzoylbenzene 3
B N N F F B N N F F B N N F F B N N F F B N N F F B N N F F B N N F F B N N F F Ph Ph

Relative singlet oxygen quantum yield measurements of photosensitizers

As mentioned in the previous section, generation of singlet oxygen can be measured effectively via DPBF degradation assay. This was performed as described in the following section.

3.2.1.

Singlet oxygen measurements of meso-substituted dibenzihomoporphyrins Bench-stable di(p/m-benzi)homoporphyrins were synthesized by Dr. Ganapathi Emandi through acid-catalyzed condensation of dipyrrole derivatives with aryl aldehydes. Coremodified expanded porphyrins have been actively investigated for application as photosensitizers. [16] These compounds are shown in Figure 3.6. The potency of derivatives 3.31 and 3.32 is likely due to the electronic contributions of the fluorine atom. For derivative 3.35 with anthracene, singlet oxygen production is potentially enhanced by this moiety which has been reported to form endoperoxides under irradiation with light with an enhanced singlet oxygen quantum yield. [18] The related macrocycles of such derivatives have not been reported to be active for DPBF degradation assay. Thus, this study is the first one to establishes the basis of these macrocycles for exploration of potential in PDT.

Singlet oxygen measurements with BODIPY-appended tetraphenylethene (TPE)

Novel tetra-BODIPY-appended TPE derivatives (BODIPY-TPE) illustrated in Figure 3.10 were synthesized, each with differences in spacing between the TPE and BODIPY cores to study their aggregation-induced emission (AIE) properties. The conjugates were studied for their efficiency to produce singlet oxygen species ( 1 O2) when measured via DPBF degradation assay as described. Hence, these molecules have a potential activity as PSs for PDT. [19] The Table 3.3 shows the singlet oxygen quantum yield for BODIPY-TPE derivatives; these are adequate as photosensitizers utilized for PDT. . Compound 3.37 had a relative singlet oxygen yield smaller than compound 3. 38 The introduction of an additional acetylene spacer and the corresponding increase in conjugation presumably leads to this enhanced yield. [20] BODIPY-TPE derivatives Singlet oxygen quantum yield (ΦΔ)

Singlet oxygen measurements of brominated porphyrins

Previous studies have reported that the addition of a bromine substituent can have profound effects on the photodynamic reactivity of porphyrins as compared to the nonbrominated parent molecule. [21] PS associated singlet-oxygen generation benefits from exploitation of the heavy-atom effect, in which the presence of a higher molecular weight atom enhances spin-forbidden processes. Photosensitizers based on similar porphyrins with different bromine atom substituent placement are therefore expected to show varying singlet-oxygen generation profiles. [22] A series of brominated porphyrin derivatives (3.39-3.41) as shown in Figure 3.13 were synthesized as potential PSs. From the data in Table 6, we can see that the brominated porphyrins are each effective PS species, comparable to (for 3.40) or exceeding (3.39 and 3.41) the H2TPP reference. Given the relative stability of the Br atom, and the ease with which this can be introduced to the porphyrin skeleton, Br substitution is a highly effective strategy for PS enhancement.

Brominated porphyrins

Conclusion

In conclusion, the efficacy of photosensitizers to be utilized in PDT or aPDT application can be evaluated via chemical assays such as the DPBF degradation assay giving a preliminary insight into the production of ROS species, an important element for an effective applied PDT. These assays provide reference for relative quantification of The synthesis of difluoroborondipyrromethene (4,4-difluoro-4-bora-3a,4a-diaza-sindacene, BODIPY) dyes was first reported by Treibs and Keuzer in 1968. [1] In the 1990s the potency and stability of these dyes as fluorescence markers and in tunable LASERs was reported; this attracted much scientific attention to this class of organic compounds. [2] With strong absorption and tunable fluorescence, coupled with the ease of synthesis of a library of these molecules, these compounds have found diverse biomedical applications. [3] BODIPY dyes are part of the class of dipyrrin fluorophores, a class of generally visibleactive organic dyes with a modifiable periphery and heterocyclic skeleton providing a simple η 2 chelate of nitrogen atoms with a single anionic charge. [4] The characteristic features of these dyes includes chemical robustness, high thermal and photochemical stability, absorption and fluorescence bands in the visible region around 500 nm, high molar extinction coefficients and high fluorescence quantum yields (φF). [5,6] The structure of BODIPY (4.1) dyes features a skeleton of a dipyrromethene ligand, with two pyrrole units linked via a methine bridge, chelating a BF2 moiety. BODIPY dyes do not obey Hückel's aromaticity rule (4n+2 𝜋 electrons). The IUPAC numbering system of BODIPY core shows eight functional positions as shown in Figure 4.1.

Synthetic methodologies

The chemistry of BODIPY dyes can be potentially tuned to obtain the desired antimicrobial activity; the simplest approach being the attachment of a pyridinium derivative. [7] The formation of a library of compounds related to this motif, including many N-heterocycles, allows understanding the relevant parameters of this chemical landscape (i.e., the development of a quantitative structure-activity relationship). [8] Dipyrromethene ligands can be prepared by using pyrroles and a condensation partner, generally an aldehyde, then subsequently complexing with BF 2 in the presence of a base. This procedure affords a BODIPY in reasonable yields after workup. [9] 4.2.1.

Strategies to synthesize BODIPY dyes

BODIPY compounds can be formed by combining pyrrole (4.2) and acyl chloride (4.3)

as shown in Scheme 4.1. [10] The coupling of these two reactants produces an intermediate acylpyrrole which cannot be isolated and is further reacted with another pyrrole to afford dipyrromethene (4.4). Addition of boron trifluoride (BF3•Et2O) under basic conditions yields the final BODIPY adduct (4.5). This approach has been used conventionally to synthesize meso-alkyl-substituted BODIPYs where an alkyl aldehyde may be unreactive. The more versatile and commonly used procedure to synthesize BODIPYs is a one-pot synthetic route as illustrated in Scheme 4.2. This occurs via an acid-catalyzed (typically trifluoroacetic acid) condensation reaction of an aromatic aldehyde (4.6) with two equivalents of the pyrrole (4.7), resulting in the formation of dipyrromethane molecule (4.8). [11] This moiety is relatively unstable towards light, air, or acid; it reacts readily with This second reaction strategy is used in this chapter as it provides an easy pathway to synthesize BODIPY derivatives with uncharged N-heterocycle units as meso substituents that can be further functionalized. These dyes are expected to be robust and allow many post-synthetic modifications, principally substitution at the N-position of the appended heterocycle and halogenation/coupling reactions at the pyrrole. The functionalization diversity obtained following this synthetic route and subsequent functionalization is the reason we used this method of synthesis to obtain a library of substituted BODIPY scaffolds with different functional groups.

N N R 4 R 1 R 3 R 2 R 3 R 2 R 1 B F F Base BF 3 .Et 2 O N HN R 4 R 1 R 3 R 2 R 3 R 2 R 1 N R 3 R 2 R 1 H R 4 O Cl CH 2 Cl 2 H + R 1 , R 2 , R 3 , R 4 =alkyl 4
Scheme 4.2. Synthetic route to meso-substituted BODIPY (4.10) dyes using aromatic aldehyde (4.6)

and pyrrole (4.7).

Derivatization of the BODIPY framework

The relative ease of synthesis and functionalization of BODIPY dyes is due to electron deficiency, which allows chemical modifications to often proceed under less forcing conditions than other aromatics. A rich chemistry of functionalized BODIPY compounds with different linkers, receptors, and several important organic molecules or bioconjugates is known, via different types of reactions. [12] NH HN O Possible modifications for the BODIPY core are as follows:

H N R 3 R 2 R 1 R 3 R 2 R 1 R 1 R 2 R 3 H R R H + N N R 1 R 3 R 2 R 3 R 2 R 1 B R F F DDQ Base BF 3 .Et 2 O N HN R 1 R 3 R 2 R 3 R 2 R 1 R 4
i) Electrophilic substitutions at the 2, 6-positions. [12][13][14] The 2,6-positions of the BODIPY skeleton, when unsubstituted, can undergo electrophilic aromatic substitution reactions akin to those encountered for aryl units.

Water-soluble sulfonate groups can be appended to the hydrophobic core of BODIPY dye. This modification does not influence the optical properties of the BODIPY core, making these modifications useful for biological applications. Furthermore, the introduction of heavy atoms such as halogens (usually Br or I) causes a bathochromic shift in absorption and emission bands which quenches the fluorescence intensity relative to the parent dye.

ii) Nucleophilic substitution at the 3, 5-positions. [12,[15][16][17] Halogens such as chlorine or iodine are good leaving groups; due to the electrondeficient BODIPY core, a 3,5-dimethyl BODIPY readily undergo nucleophilic substitution reactions. Several nucleophiles can be used, forming carbon to carbon, nitrogen, oxygen, or sulfur bonds, adding the advantage of possible extended conjugation, enabling a bathochromic shift in the spectrum. The 3,5-positions are reactive for palladium mediated cross-coupling reactions such as Sonogoshira coupling, Suzuki coupling and Heck coupling. This allows the incorporation of functional groups such as ethynyl, and aryl-substituents to form long-wavelength BODIPY-based fluorescent biosensing materials and bio-labels. [12] iii) Functionalization at the C-8 position. [18][19][20] The C-8 position of the BODIPY core has been used for the incorporation of molecules such as ion capture ligands, water solubilizing groups, donor-acceptor groups, and biomolecules. This modification site is the most versatile, and has been used widely to produce molecules, such as the BODIPY-anthracene dyads (BADs). In the context of this manuscript, this strategy was utilized to make several molecules active for antimicrobial photodynamic therapy. iv) Replacement of the meso-carbon with nitrogen to form aza-BODIPY dyes. [18,21,22] A class of dyes related to the BODIPY by replacement of the C8 atom with nitrogen, named aza-BODIPYs, can be formed via Michael addition reactions or via the reaction of phthalonitrile and arlymagnesium bromides. The introduction of an electronegative nitrogen atom affects the HOMO-LUMO energy gap through stabilization of the LUMO causing a bathochromic spectral shift. This moiety has been used as a promising fluorophore for in vivo molecular imaging and theragnostic applications.

v) Fusion of aromatic rings to the pyrrole units. [23][24][25] Fusing aromatic rings to pyrrole precursors (e.g., indole or isoindole) causes red-shifted absorption and emission bands of the synthesized BODIPY dyes. Strategies such as the use of aryl-fused pyrroles or retro Diels-Alder syntheses of norbornane-derived pyrroles can be used to derive these extensively π-conjugated systems. These BODIPY dyes have found extensive use in molecular imaging. [23] vi) Nucleophilic substitution of the fluorine at the boron center. [14] The replacement of the fluorine atoms by, e.g., ethynyl or oxyanion nucleophiles has been performed to derivatize BODIPY dyes with enhanced photostability, forming highly luminescent redox-active complexes. Substitution of the fluorine atoms generally employs the use of organometallic reagents, typically an organolithium or Grignard complex.

The BODIPY dyes synthesized via each of these post-synthetic functionalization methods shift the emission maximum to the 510-800 nm range; those with crowded substitution patterns have additional advantages of having higher extinction coefficients (ε > 50,000), enhanced photo-and chemical stability and insensitivity towards the solvent polarity. [26,27] It has also been shown that electrochemical properties of the BODIPYs can be tuned followed by modification on the pyrrolic core; these tuning strategies of the BODIPY core have enhanced the utility of this molecule in fields of cell imaging, biolabeling or as chemical sensors as multiple chemically similar but photochemically distinct labels can be employed simultaneously. [28,29] With the versality of this molecule well established, it is important to note the most common drawback encountered, the aggregation and induced quenching of florescence in aqueous media. [29] Strategies to avert aggregation include introduction of static charge to introduce repulsion and use of drug-delivery systems; these strategies are both employed in this investigation to enhance utilization in biological strata most importantly in aPDT.

Applications of BODIPY dyes

As previously stated, BODIPY dyes have been studied and explored for their applications since the early 1990s, as a readily accessible, versatile, biocompatible, and stable fluorophore. [30] Researchers have explored the use of such dyes in molecular biolabeling, cell imaging, chemical sensing, solar cells, and two-photon absorption. [31] among other emerging uses. In the last decade, the BODIPY dyes have been reported

to be potential PS in aPDT, though few of examples of such dyes designed for this purpose exist in literature. The current state of research is summarized below.

BODIPY dyes in antimicrobial photodynamic therapy (aPDT)

Obviously, for a PS active for aPDT it is necessary that the PS be soluble in water. Often by using specific solubilizing functionalities a charged group introduced to the molecular backbone can be provided to ensure a good degree of interaction between the bacterial cell wall and the PS itself. [32] . Several dyes based on porphyrin and similar macrocycles possessing these characteristics are typically rigid and planar structures at their core, and have large, conjugated surface. [33] However, a disadvantage to using such dyes is the weak absorption in the visible region, a lack of target specificity, and a tendency to aggregate at high concentrations. [34][35] Some research indicates that an extension of the π-conjugation through substitution with styryl, aryl and ethynyl aryl substituents at the 1, 3, 5, and/or 7-positions or aromatic ring fusion, or formation of aza-BODIPY formation, etc. can significantly enhance and red-shift the absorption band, allowing overlap with the optical window for tissue penetration in the 650-1000 nm region. [36] . The incorporation of heavy atoms such as halogens to the BODIPY dye enhances the rate of intersystem crossing to the triplet state, which is generally low in BODIPY dyes, thus enabling the production of singlet oxygen by decreasing the fluorescence quantum yield. [37] In addition, a few watersoluble BODIPY derivatives have been synthesized by functionalization with sulfonic groups, phosphonates, sulfonated peptide chains or oligo-ethylene glycol chains. [38]

Examples of BODIPY dyes used in aPDT

To generate efficient BODIPY PS for effective aPDT it is important to employ all the above modifications simultaneously, to synthesize red-wavelength BODIPY photosensitizers (i.e., those that absorb > 600 nm) with improved singlet oxygen production, enhanced water solubility and effective cell interaction and penetration. A difficult task, steps toward this ideal PS have been taken by the addition of cationic charges to the BODIPY dye or the exploitation of the heavy atom effect. [39] The first report of aPDT efficiency of BODIPY dye was published in 2012 by Orlandi and coworkers wherein they synthesized BODIPY dyes with a meso-pyridinium group and 2,6-disubstituted with iodine atoms as shown in Figure 2. [40] These dyes (4.11 and 4.12)

showed efficacy against a broad spectrum of microbial species (S. xylosus and E. coli) as shown in Table 4.1. BODIPY dyes 4.12 also an efficient inhibition of growth bacterial biofilm of Gram-negative P. aeruginosa. This evidences the need for the introduction of cationic charges for increased interaction with the cell membrane of the target microbe. The heavy atom effect via halogen addition is very important, as this can enhance the spin-forbidden process which enhances the aPDT efficiency of the BODIPY dyes [44] . Some examples of this are illustrated in where a series of mono-and di-halogenated BODIPYs bearing electron-donating substituents have been described. [41] They showed effective aPDT against the Grampositive species of B. subtilis and Gram-negative species of E. coli. Another report of such heavy atom effects showed for BODIPYS with meso-acetoxymethyl moieties and substituted at the 2,6-positions with bromine or iodine as shown in Figure 3b. Both dyes could inactivate the Gram-negative species of E. coli and the BODIPY dye with bromine (4.23) was more efficient than the one substituted with iodine (4.24). [42] A hexa-brominated BODIPY derivative with a meso-aniline substituent conjugated to Au nanorods, as shown in quantum yield (ΦΔ) enhanced from 0.64 of the parent BODIPY (4.25) to 0.76 upon conjugation with 4-AuNR (4.26), both very high values due to the heavy atom effect. [43] shown in Figure 4.5 displayed a significant phototoxic assay as against the Grampositive S. aureus and Gram-negative P. aeruginosa. [44] A very effective inactivation of both these strains was observed by dibrominated thiol derivatives (4.34-4.

BODIPY scaffolds synthesized

Throughout the manuscript it was emphasized how important it is that a molecule to be used in biological research has a strong interaction with the microbial cells, along with an enhanced solubility in biological medium. The BODIPY dyes have long been established to have remarkably high quantum yield for singlet oxygen production along with good photostability. [14] However, as is often the case with large π-system containing molecules, poor interactions with cell structures and limited solubility hampers their use as biological probes. Also, BODIPYs are known to have high fluorescence quantum yields and thus deactivates the singlet excited state through ISC. This reduces the formation of production of 1 O2 (ΦΔ) species. Therefore, it becomes of utmost importance to develop molecules which can overcome these limitations for aPDT.

Synthesis of the described BODIPY compounds have the apart from being time-efficient, has an added advantage of being a convergent approach-that one heterocycle-bearing precursor can be the starting material to innumerate final compounds. Hence a library of BODIPY dyes was synthesized with meso-aryl-substitutions which can be postfunctionalized with moieties improving their potential for aPDT. The first functionalization strategy was to introduce ionizable substituents on a N-heteroatom of the mesoheterocycle or by replacement of fluorides on boron in the 4-position via N-alkyne-Grignard reagents making these dyes charged water-soluble derivatives for enhanced aqueous solubilization. [45] The second strategy used in this chapter was to introduce heavy atoms by halogenation at the 2,6-positions to enhance the photophysical and cytotoxic activity against bacteria. Finally, extended π-conjugates of these molecules were synthesized via double Sonogoshira cross-coupling reaction via 2-position postfunctionalization enhances the optical window of the compounds towards the bathochromic shifts and hence increases the usability of these scaffolds for deep tissue treatments. [40] 4.5. Results and discussion

Synthesis of aPDT active BODIPY dyes

meso-N-heterocyclic BODIPY

The ease of synthesizing this scaffold from aryl aldehydes via condensation with two pyrroles affords the simplest and a direct route to add functionality and derive diverse derivatives of the BODIPY fluorophore. Following the classic three-step, one-pot strategy was employed in the BODIPY synthesis as shown in Scheme 4.

Water-soluble BODIPYs

The meso-N-aryl BODIPY derivatives (4.46, 4.54, 4.48 and 4.56) described in the previous sections offer reactive points for post-functionalization with ionizable groups to enhance the utilization window of such derivatives in aPDT due to improved membrane interactions and solubility. [55,56] The first examples to be prepared with this strategy were obtained via a facile one-step synthesis of methyl iodide (4.62) functionalized derivatives 4.46a, 4.54a, 4.48a and 4.56a, respectively, as illustrated in Scheme 4.4. Another strategy to prepare water-soluble derivatives of the corresponding BODIPYs is to synthesize boron-ethynyl-substituted carboxylate derivative. [45] A two-step synthesis To synthesize the BODIPY derivatives 4.46d and 4.50a, the dimethylamino moieties were introduced onto the boron atom via replacement of the fluoride atoms with the Grignard reagent 1-(N,N-dimethylamino)-prop-2-yne (4.64) to synthesize derivatives 26 and 27 in yields of 30-45%. Then an attempt to quaternate these scaffolds with 4bromomethyl benzoic acid was done. [45] The reaction did not yield the expected product but a mixture of compounds which were difficult to separate and isolate due to lack of solubility in organic solvents.

Target charged water-soluble derivatives are obtained as orange, or deep red crystals in 60 to 85% yields as illustrated in Table 4.3.

Heavy-atom derivatives of BODIPYs

The 2,6-positions of the BODIPY core have known to readily undergo a nucleophilic attack by halide ions such as iodide. [40] This knowledge in hand, BODIPYs ( This was achieved by performing first Sonogashira cross-coupling of derivative 4.46e

with trimethylsilyl acetylene (TMSA) and derivative 4.46f was obtained and purified with a 78% yield. This derivative was then deprotected (BODIPY 4.46g) -50 ºC in the presence of TBAF and a pink solid was obtained and purified in 85% yield. The molecule was then subjected to a second Sonogoshira cross-coupling; however, the cross-coupling reaction did not proceed as expected and only a homo-coupled product (4.66) was isolated 42% yield. Some difficulties in the purification of 4.66 were met, due to the poor solubility of this derivative in common solvents. 1 H NMR revealed the structural details of the complex but was unspecific as to the nature of the bridge, which was confirmed with elucidation of the X-ray crystal structure. TMS derivative 4.51a was also obtained in 65% yield following a similar procedure as described above from compound 4.51b; dimerization of this compound into a coupled derivative using the same procedure as above was unsuccessful.

The synthesized library of differentially functionalized BODIPYs allowed for the investigation for effect of such functionalization on the photophysical and cytotoxic

X-ray Crystal determination

Crystallographic structural elucidation of BODIPY dyes

Crystal growth is a crucial step in characterizing a new molecular compound -a good quality crystal allows the determination of the structural details of the desired compound through X-ray diffraction. [46] A crystalline form of a drug affects properties such as its bioavailability, stability, solubility, dissolution rate, and bioavailability, and the crystalline state of a drug is crucial to understanding the pharmacology and is a critical step in commercialization through patent application. [47] The crystal engineering of organic molecules is an emerging field focused mainly on the identification and control of intermolecular and supramolecular features to yield a designed solid-state conformation or packing.

Our research group has been using BODIPY and related compounds for applications ranging from photo up-conversion to photosynthesis, incorporated into hydrogels and scaffolds. [48,49] Detailed X-ray studies on the structures of a range of BODIPY compounds has given insight into their photophysical properties; It has been shown that the conformation of the BODIPY has an effect on the measured photophysical properties of the molecule. [50] 1618 BODIPYs have been described and characterized in the crystallographic solid phenanthroline among many others. [51,52] No obvious structural differences in the BODIPY core are apparent from the inclusion of different aryl or aromatic units.

Inclusion of quaternary N-containing heterocycles in this position is rare, with only a few examples reported, and significant differences from the photophysical properties of the parent, neutral compounds owing to differences in the extent of the aromatic unit. These heterocycles can provide one half of a 'push-pull' architecture. [53] The structural characteristics of BODIPY compounds have been previously described by a combination of bond distances, plane-to-plane angles, as well as displacements from mean planes. Compounds' photophysical attributes are similarly responsive to atom substitutions and ionic charges, aromaticity, and functional group, as well as induced intermolecular interactions and solvatochromic effects. While the structural minutiae may be observed in the solid-state, this observation is generally only of one conformational energetic minima of the electronic ground state; intuition is required to extrapolate from an individual snapshot.

X-ray crystallography of representative compounds allows for insight into the internal bonding, conformational shape, and molecular packing of compounds. Generally, crystals of these compounds were grown by slow evaporation of methanol or dichloromethane solution as described in the experimental section. The structure solution in each case corresponded to the indicated compound, in agreement with spectroscopic results.

Each of the molecules discussed here features the 1,3,5,7-tetramethyl substitution pattern on the BODIPY core; this should negatively influence the ability of the compound to approach coplanarity with the aryl ring or to delocalize the aryl charge to the BODIPY component. Thus, we expected the influence on the BODIPY core of different nitrogen-containing heterocycles, and therefore photophysical changes within this subset, to be minimal. Nonetheless, the influence of charge will dramatically influence solubility and likely solvatochromic effects.

meso-Aryl BODIPY derivatives

The single crystal X-ray crystallographic studies of compounds were kindly performed by Dr. Christopher J. Kingsbury Generally, the introduction of the N-heterocycle H or X-bond acceptor motif (formally a nitrogen with lone pair, e.g., pyridine, 2-and 4-quinoline, acridine, pyrimidine) in these compounds demonstrates at least one non-negligible intermolecular interaction (e.g., C-H⋯N) around the van der Waals limit, similar to many aromatics. [58] This, however, is usually confounded with the C-H⋯F interactions in which these molecules are engaged. The aggregation of individual molecules in the solid state (i.e., thermodynamic equilibrium) can often give an idea as to the dominant preferred interactions of these molecules in solution state or on surfaces. The molecules depicted above do not form strong dimeric arrangements in the solid state, and therefore would be unlikely to do so in a good solvent.

An example of these interactions is shown in

Water-soluble compounds

The quaternarization of the dorsal N-heterocycle on the BODIPY was our means to provide a charged group for heightened antimicrobial response. The introduction of charge has additional effects in the structural chemistry of these compounds, providing a charge separation, with additional anions to consider alongside the bond-order effects of pyridinium-based compounds. [59] The structures of five compounds, each with a quaternary N-heterocycle on the 8position of a BODIPY, are presented in Figures 4.12 The five compounds reported here each exhibit the expected BODIPY structure, with pyridyl and quinolinyl rings with similar plane-to-plane angles with the BODIPY system as in their neutral counterparts, as seen in Table 4.5. if the compound had any doublebond character, we would have seen shortening of the C-C distance and a movement towards coplanarity as compared to a neutral molecule; the slight lengthening of this bond and variable angle are indications that this behavior is not occurring.

BODIPYs with extended π-conjugated systems

The single crystal X-ray crystallographic studies of the compounds in this section were kindly performed by Dr. Brenden Twamley. As described in the synthesis section, the formation of compounds with two BODIPY units introduces extended conjugation and to be expected due to similar distances in the bridge. [61] A selection of the intermolecular interactions engaged by this compound are demonstrated in

Non-BODIPY Structures

The single crystal X-ray crystallographic studies of compounds were kindly performed by Dr. Christopher J. Kingsbury. Two crystal structures were collected during this study which did not include the BODIPY motif and are described here. The formation of the 1,2,5,7-tetramethyl-2,6-diethyl-8-(2-thiazolyl)-4-bora-3a,4a-diaza-s-indacene (4.52a) resulted in a side-product which was visibly iridescent and crystalline upon evaporation of the column fragment. The structure shows a tripyrrin-like arrangement of N-atoms and heterocycles -but with C-for-S replacement on the periphery. The structure of this compound is shown in Figure 4.17. This crystal consists entirely of the Z-Z-cis-cisisomer, implying that the formation of the internal three-center H-bonding arrangement is a favorable conformational driver for this species. Investigating the potential N-to-S switchable metal coordination of this proligand is an interesting avenue for future research, potentially acting as a C-bridged-mimic of the activity of bis(2thiazolylimino)isoindoline investigated as a potential ligand for catalysis. [62,63] a) b) c) d)

Spectroscopic investigations

The UV-vis spectroscopic properties of BODIPY dyes were investigated in dichloromethane and methanol (water-soluble BODIPY derivatives). The results are summarized in tetramethyl-) substitutions as shown in Figure 4.18, with a λmax shift of 24 nm for the later. The UV-vis absorption spectra for water-soluble derivatives does not show any significant change of absorption maxima from the parent meso-aryl-BODIPYs, implying that the core of BODIPY is unaffected by this process. The position of the absorption maximum was sensitive to the substituents in the 2position as illustrated in Table 4.7. The attachment of a bromo or iodo substituent is known to induce a significant red shift of absorption maxima of between 25 and 30 nm, respectively, due to electrostatic (withdrawing) contribution to the π-system of the BODIPY core (Table 4.7). [40] Similar compared to the parent BODIPY 4.51. The 2-position TMS-functionalized and TMSdeprotected derivatives also show a similar shift as the iodinated parent BODIPY in absorption spectra due to ligand-based π-π* transitions. [40] The UV-vis evaluation of TMS deprotected derivative 4.46g when compared with coupled derivative 4.66 shows a shift in absorption maxima of 33 nm (Table 4.7), as expected for the increased conjugation across the butadiyne bridge as shown in Figure 4.20. This wavelength shift increases the therapeutic window of such derivative to be utilized in aPDT, should the solubility issues be able to be overcome.

functionalization

Singlet Oxygen Generation Studies

In chapter 3 we explained how important it is to evaluate the singlet oxygen production of our PSs to get an insight to the activity of these molecules for real world aPDT.

Synthesized and post-functionalized BODIPYs were tested for production of singlet oxygen species using standard DPBF assay, like that used in Chapter 3. Each photosensitizer was referenced to an equivalent concentration of Rose Bengal (RB) (ΦΔ = 0.76) (UV absorbance, RB (0.15 mM) in DCM). [64] (Figure 4.21). The UV-absorbance degradation graphs of meso-aryl derivatives of BODIPYs are illustrated in Figure 4.22 and 4.23 and Table 4.8. Singlet oxygen quantum yields ranged from 0.007 to 0.77 (shown in Table 4.8 and 4.9). meso-Aryl BODIPYs 4.52, 4.55, 4.58 and 4.60 have been identified to be moderate singlet oxygen generators (ΦΔ = 0.5-0.3). Along with their key subcellular localization, such generation leads them to possess highly effective photosensitizer qualities. [65] Iodinated derivatives (4.46e, 4.49a and 4.51a) show high singlet oxygen quantum yields (∼ΦΔ = 0.7) which is subsequently correlated to their inhibition efficiency as reported in next section. Derivative 4.53a also inhibited the growth of P. aeruginosa at <100µM (MIC/MBC).

BODIPYs

These results could be explained with earlier arguments of induced heavy-atom effect of such derivatives which forms a long-lived, high-yield triplet excited state for an efficient singlet oxygen production, thus leading to an enhanced aPDT action of the dye.

Water-soluble derivatives 4.48a and 4.54c displayed an exceptional inhibition S. aureus at <30µM (MIC/MBC), this could be due to charge aided interaction with the bacteria and enhancement in solubility across the bio-membranes of such PSs. Surprisingly derivatives 4.48a, 4.56a and 4.53a also display dark toxicity assay which can be attributed to aggregation of these derivatives under the reaction conditions, hence these attributes need to be resolved to put in maximum output of such PSs against the microbes.

Conclusion and future perspectives

A very few examples of such a dynamic moiety, BODIPY dye has been reported in literature for their application in aPDT. With possibility of functionalizing the BODIPY on its core with different and specialized functional groups opens a window of utilization of these molecules against the microbes. To achieve this goal a series of photo-stable meso-aryl-BODIPYs and their derivatives bearing charged water-solubilizing groups, iodinated derivatives and conjugated dyes were synthesized in this chapter. These derivatives were synthesized with an aim to enhance the antimicrobial therapeutic window of BODIPY scaffold. Use of different synthetic and post-functionalization strategies gave a library of 31 novel BODIPYs. NMR characterizations and UV spectrometry of the synthesized BODIPY revealed the difference in resonance of corresponding protons and the shifts in absorption maxima corresponding to postfunctionalization of the parent BODIPY core. With introduction of heavy atoms in the BODIPY core itself, the UV absorption maxima shifts to about 25-30 nm which is in agreement with literature reported derivatives and thus with this the optical window of these compounds expands against a wider strata of microbes and for treatment of deep tissue infections. [40] Similarly for the extended π-conjugated systems the shifts for absorption maxima correlates to literature reported compounds which on increase in conjugation undergo bathochromic shifts making their utilization for treatment of deepseated infections more effective. [40] Different functionalization strategies and substitutions of BODIPY scaffolds affected the singlet oxygen production as measured via the DPBF degradation assays and the inhibition assays tested against the Grambacteria. Production of singlet oxygen species (ΦΔ) with DPBF degradation assay showed good results for the BODIPY derivatives specially for 2-position nucleophilic iodide ion substitution which can be correlated with the enhanced ISC, a phenomenon correlating with introduction of the heavy atom in PSs. [40] Antimicrobial and biofilm inhibition assays of Gram-strains of bacteria revealed more details of efficacy of the scaffolds and these evaluations revealed that some of these post-functionalized and substituted scaffolds exhibit enhanced activities. These results can also be correlated to literature reported scaffolds where the post-functionalization strategies as described above with water-soluble groups and heavy atom introduction increases the efficacy of molecules against the microbes making these PSs an ideal candidate for aPDT. [40] Thus, we can conclude from this chapter that with the versatility of the BODIPY molecule, these molecules could be utilized to their utmost potential following different synthetic and post-functionalization routes. Hence, this opens a window of exploration into future of aPDT with easy to make and chemically modifiable BODIPY and related dyes.

For future work related to this manuscript, these BODIPY dyes can possibly be formulated with a drug delivery platform as described in the previous chapter 1 and 2 via ionic interaction or covalent linkages and could be evaluated for their aPDT potential as a PS-biopolymer formulation with improved solubility, biocompatibility and enhanced interaction with the bacterial populations which could possibly enhance the aPDT potential as compared to their parent BODIPY molecules.

We have been preparing cellulose nanocrystals (CNCs), biopolymeric drug carriers to form a drug carrier platform for conjugated BODIPYs. For this CNCs have been oxidized

under reaction with sodium hypochlorite, sodium bromide and TEMPO at pH=10 to obtain carboxylate functionalized CNCs as illustrated below in Figure 4.30. [67]

Material characterization

The freeze-dried conjugates were analyzed by scanning electron microscopy (SEM) General Procedure A: PS-hyaluronate conjugate (2.25-2.26): Hyaluronic acid (100 mg) was dissolved in 20 mL of distilled water at a concentration of 5 mg. mL -1 . PS (30 mg) with 1 M ratios of HA repeating unit was added to the HA solution. After that, EDC•HCl (2 eq.) and sulfo-NHS (2 eq.) were added to activate the carboxyl groups of HA. The pH of the mixed solution was adjusted to pH 4.7 using 1 N NaOH and 1 N HCl. about 1.5 h at 82 °C. The resulting solution was dried over anhydrous NaSO4 and evaporated in a rotavapor. The residue was purified by washing the resulting solid with ethyl acetate to obtain the pure product as a red crystalline solid.

General Procedure E: BODIPYs (4.46b, 4.54b, 4.48b and 4.56b): Previously synthesized BODIPY (1 eq.) and 4-(bromomethyl)benzoic acid (8 eq.) were dissolved in anhydrous dry toluene (4 mL) and refluxed for about 18 h at 111 °C. The resulting solution is dried over anhydrous sodium sulphate and evaporated in a rotavapor. The residue was purified by washing the resulting solid with ethyl acetate to obtain the pure product as a red crystalline solid.

General Procedure F: BODIPYs (4.46b, 4.54b): Previously synthesized BODIPY (1 eq.) and 4-bromobutyl phenyl ether (8 eq.) were dissolved in anhydrous dry toluene (4mL) and refluxed for about 18 h at 111 °C. The resulting solution is dried over anhydrous sodium sulphate and evaporated in a rotavapor. The residue was purified by washing the resulting solid with ethyl acetate to obtain the pure product as a red crystalline solid.

General Procedure G: BODIPY (4.46d and 4.50a): 3-Dimethylamino-1-propyne (4.5 eq.) was dissolved in dry THF (8 mL) at room temperature under argon. After 10 min n-BuLi in Hexane (4 eq.) was added dropwise and the reaction was stirred for 45 min under inert atmosphere of argon. BODIPY (1 eq.) was dissolved in dry THF and was then added to the above reaction dropwise and the reaction was refluxed for 2.5 hat 66 ºC. The reaction mixture was quenched with deionized water (20 mL) and was then filtered and washed with a solution of saturated NaHCO3 (2 x 50 mL) followed by washing with deionized water (2 x 50 mL). The resulting solution is dried over anhydrous sodium sulphate and evaporated in a rotavapor. The residue was purified by flash column chromatography eluting with DCM:MeOH (2:1) to yield the pure product as an orange-green crystalline solid.

General Procedure H: BODIPYs (4.46e, 4.49a, 4.51a and 4.53a): Previously synthesized BODIPY (1 eq.) and N-iodosuccinimide (1 eq.) were dissolved in 10 mL of DCM at room temperature. The reaction mixture was stirred at room temperature for 30 min and N-iodosuccinimide (0.25 eq.) of was added twice at an interval of 40 min. The

Ch 2: Formulating photosensitizer-conjugates and their antimicrobial evaluation

Purpurin-18 (2.17)

Purpurin-18 (2.17) was extracted and synthesized from Spirulina maxima powder (50 g) was used to extract pigments with acetone (1 L) followed by addition of 6 M NaOH (200 mL) and 100 mL concentrated HCl. Purification by column chromatography (silica gel;

CHCl3/EtOH, 8:2, v/v) and removal of solvents followed by recrystallization (CHCl3/petroleum ether) to yield a purple solid. Analytical data is identical to the literature. [1,2] Rf = 0.27 (CHCl3/EtOH: 96/4); 1 H NMR (CDCl3): δ (ppm) = 9.54, 9. 4.39 (q, J = 6.0 Hz, 1H, 18-H); 3.72 (s, 3H, 12-CH3); 3.59 (q, J = 6.6 Hz, 2H, 81-CH2);

Chlorin p6-PEI (2.18)

In accordance with previously used procedure by Sol lab to obtain chlorin p6-PEI (2.18) using purpurin-18 (2.17) (100mg, 1equiv.) PEI (polyethyleneimine) (600 Da) (98.1mg, 1equiv.) in 100 mL chloroform to yield a dark-green solid (98.1% yield). Analytical data is identical to the literature. [1] M.p. = >300°C; UV-Vis (CH2Cl2) λmax (ε [x10 3 M -1 cm -1 ]) = 400 (52.4); 502 (6.0); 530 (2.8); 552 (1.9); 611 (2.4); 665 (

2.24

In accordance with general procedure porphyrin 2.24 was synthesized as described above using porphyrin 3 (50 mg, 0.30 mmol), iodomethane (0.24 mL, 3.96 mmol) and anhydrous DMF (10 mL) as starting material to yield a purple-brown powder (82% yield).

Analytical data is identical to the literature. DLS analyses and zeta potential analyses of conjugates: 2.25 has a diameter of 342 ± 52.8 nm and zeta potential of -34 mV.

UV-vis analysis:

The relative molar percentage of photosensitizers in the conjugate structure was determined based on monomer concentration to be 3% for this conjugate.

UV-vis analysis:

The relative molar percentage of photosensitizers in the conjugate structure was determined based on monomer concentration to be 18% for this conjugate. In accordance with general procedure B as described above using 2,4-dimethylpyrrole (2.0 mL, 19.4 mmol) and 4-pyridine carboxaldehyde (0.84 mL, 9 mmol) and final product was obtained and purified by column chromatography (silica gel; DCM/Hex, 6:4, v/v) and removal of solvents followed by recrystallization (CHCl3/Hex) to yield a green solid (16% yield). Analytical data is identical to the literature. In accordance with general procedure B as described above using 2,4-dimethylpyrrole (2.0 mL, 19.4 mmol) and 5-pyrimidine carboxaldehyde (0.97 g, 9 mmol) and final product was obtained and purified by column chromatography (silica gel; DCM/Hex, 8:2, v/v) and removal of solvents followed by recrystallization (DCM/Hex) to yield green solid (8% In accordance with general procedure B as described above using 2,4-dimethylpyrrole (2.0 mL, 19.4 mmol) and 1-methyl-2-imidazol carboxaldehyde (0.99 g, 9 mmol) and final product was obtained and purified by column chromatography (silica gel; DCM/Ethyl Acetate, 9:1, v/v) and removal of solvents followed by recrystallization (DCM/Hex) to yield green solid (8% yield). M.p. = 175 -180 °C; Rf = 0.32 (DCM/Hex: 90/10); 1 H NMR (400 MHz, CDCl3) δ 7.19 (s, 1H, Ar-H), 7.04 (s, 1H, Ar-H), 6.01 (s, 2H, 2-H, 6-H), 3.49 (s, 3H, Ar-N-CH3), 2.54 (s, 6H, 3-CH3, 5-CH3), 1.41 (s, 6H, 1-CH3, 7-CH3). 13 In accordance with general procedure B as described above using 2,4-dimethylpyrrole (2.0 mL, 19.4 mmol) and the benzaldehyde (1.08 mL, 9 mmol) and final product was obtained and purified by column chromatography (silica gel; DCM/Hex, 1:1, v/v) and

B N N F F N N B N N F F
removal of solvents followed by recrystallization (DCM/Hex) to yield green solid (21% yield). Analytical data is identical to the literature. [5] In accordance with general procedure C as described above using 3-ethyl-2,4dimethylpyrrole (2.2 mL, 17.9 mmol) and 4-pyridine carboxaldehyde (0.84 mL, 9 mmol) and final product was obtained and purified by column chromatography (silica gel; DCM/Hex, 6:4, v/v) and removal of solvents followed by recrystallization (DCM/Hex) to yield green-red solid (15% yield). Analytical data is identical to the literature. [6] In accordance with general procedure C as described above using 3-ethyl-2,4dimethylpyrrole (2.2 mL, 17.9 mmol) and 2-thiazole carboxaldehyde (1.02 g, 9 mmol)

B N N F F N B N N F F N S
and final product was obtained and purified by column chromatography (silica gel; DCM/Ethyl acetate, 9.5:0.5, v/v) and removal of solvents followed by recrystallization (DCM/Hex) to yield green-red solid (13% yield). M.p. = >300 °C; Rf = 0.39 (DCM/Hex: 90/10); 1 H NMR (400 MHz, CDCl3) δ 7.98 (d, J = 3.2 Hz, 1H, Ar-H), 7.61 (d, J = 3.2 Hz, 1H, Ar-H), 2.53 (s, 6H, 3-CH3, 5-CH3), 2.30 (q, J = 7.5 Hz, 4H, 2-CH2, 6-CH2), 1.34 (s, 6H, 1-CH3, 7-CH3), 0.98 (t, J = 7.6 Hz, 6H, 2-CH3, 6-CH3). 13 C NMR (101 MHz, CDCl3) δ 154. 63, 136.66, 132.42, 131.61, 118.59, 17.28, 14.59, 12.55, 9.37. 19

4.59a

M.p. = 200-205 °C; 1 H NMR (400 MHz, CDCl3) δ 11.88 (s, 1H, ), 7.92 (t, J = 5.5 Hz, 2H, Ar-H), 7.13 (d, J = 3.3 Hz, 2H), 5.97 (d, J = 9.3 Hz, 2H), 2.50 (q, J = 7.6 Hz, 2H, Ar-H), 2.06 (s, 3H, Ar-H), 1.19 (t, J = 7.6 Hz, 3H, Ar-H). 13 Previously synthesized BODIPY (4.46f) (1 eq.), CuI (0.1 eq.) and Pd(PPh3)Cl2 (0.05 eq.) were dissolved in dry THF:TEA(4:1) (5ml) at room temperature under inert atmosphere of argon. After stirring for 30 min trimethylsilyl acetylene (TMSA) (10 eq.) was added under argon. The reaction mixture was refluxed for 20 h. The mixture was then filtered and washed with a solution of saturated NaHCO3 (2 x 50 ml) followed by washing with deionized water (2 x 50 ml). The resulting solution is dried over anhydrous sodium sulphate and evaporated in a rotavapor. The residue was purified by flash column chromatography eluting with Hexane:Ethyl acetate (9:1) to yield the pure product as a pinkish-orange crystalline solid (85% yield). M.p. = >300 °C; Rf = 0.69 (DCM/Hex:

Singlet oxygen measurements

The photo-irradiation of the samples were performed in quartz cuvettes (2×1×1 cm) under an irradiation via a polychromatic light source (Philips, 15V-150W lamp), equipped with a 532 nm diode pumped solid state green laser system (CW532-04, average intensity of 10 mW•cm -2 ). The temperature of the sample was maintained at 18 °C using a Peltier element (Cary Peltier 1×1 Cell Holder). Relative singlet oxygen (

Antimicrobial evaluation

General procedure for diffusion test for bacterial strains: Diffusion tests were performed through agar-tryptic soy (TSA) on Gram-positive S. aureus and Gramnegative E. coli. For PSs alone the concentration was set at 100µg/mL in TS broth, and for HA conjugates formulations with PSs the concentration was set to 350µg/ml. The stock solution (50 µL) was deposited onto each ring for each formulation and PSs. The Petri dishes were dried for 15 minutes and 2 of each inoculated Petri were kept under white LED light (total fluence: 25 J/cm²) and 2 of them were in dark condition at 37 °C.

After irradiation, the Petri dishes were incubated at 37 °C in the dark.

General procedure for MIC/MBC evaluation: Two different plates for each strain were prepared: one plate for the dark condition and a second plate for light irradiation. Two controls were included: a bacterial growth control with no molecule added and negative control with PBS without bacterial inoculum and no molecule. The microplate was incubated at 37 °C for 1h in dark conditions. After that, bacteria were irradiated with white LED light (total fluence: 25 J/cm²). During the time of irradiation, the second plate was maintained in the dark. Then, 100 µL of 2-fold concentrated TS broth were added to each well and the microplate was incubated at 37 °C for 24 h. The Minimal Inhibition Concentration (MIC) is defined as the concentration of compound in µM, where a bacterial growth was observed. Three independent experiments were performed with each strain with CP-HA and the corresponding porphyrin. potential, SEM imaging, UV-vis analysis and rheological evaluation confirming the covalent conjugation of PSs on conjugate platform (hyaluronic acid). These conjugate formulations were tested against the Gram-negative Escherichia coli and Grampositive Staphylococcus aureus bacteria for their potency for aPDT. The PS-HA conjugates with photo-bactericidal studies showed a minimal antimicrobial action in the presence of light which could be due to strong negative charges of the carboxylic acid residues of hyaluronic acid which might lead to a lower interaction between the formulation and the bacterial walls. This however could be potentially resolved with either increasing the number of PSs to be conjugated or by employing cross-linkers to accommodate the PSs within the conjugate synthesized.

General procedure for evaluation of inhibition of biofilm growth:

In against bacteria, finding that some of these molecules were able to inhibit the growth of bacteria and their biofilms under light irradiation, demonstrating the efficiency of such strategies for a potential aPDT.

Keywords: Photosensitizers, Antimicrobial photodynamic therapy (aPDT), Hyaluronic acid, BODIPY, Singlet Oxygen, Diphenylbenzofuran (DPBF)

Figure 1 . 5 .

 15 Figure 1.5. Chemical structure of Methylene Blue (1.1), Toluidine Blue (1.2), Eosin Y (1.3), Erythrosine (1.4) and Rose Bengal (1.5).

Figure 1 . 7 .

 17 Figure 1.7. Chemical structure of porphyrin (1.7).

1 . 8 .

 18 Figure 1.8. Chemical structure of TMPyP•4(I -) (1.8).

Figure 1 . 9 .

 19 Figure 1.9. Tricationic derivatives a) Tri-Py + -Me-PF (1.9) (heavy atom) and b) Tri-Py + -Me-COOH (1.10).

Figure 1 . 12 .

 112 Figure 1.12. Chemical structure of chlorin e6 (1.13).

Figure 1 . 13 .

 113 Figure 1.13. Chemical structure of hypericin (1.14), curcumin (1.15) and Cu-chlorophyllin (1.16).

  14)-mediated aPDT renders Gram-positive bacteria inactive, including

Figure 1 . 15 .

 115 Figure 1.15.Different strategies for incorporating photosensitizers in hydrogels.[120]

Figure 1 . 18 .Figure 1 . 18 .

 118118 Photosensitizer

  including P. gingivalis. The nano formulation was obtained by coupling of RB with CS with the coupling agent 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC). Biological assays were performed on the biofilms of E.

Figure 1 .

 1 Figure 1.20, enhances the solubility of PSs, making them a promising treatment option.

Figure 1 . 20 .

 120 Figure1.20. Synthesis of ZnPc-colistin-chitosan hydrogel.[158]

Figure 1 . 21 .

 121 Figure 1.21. Conventional liposomes are made of phospholipids (A); PEGylated/stealth liposomes contain a layer of polyethylene glycol (PEG) at the surface of liposomes (B); targeted liposomes contain a specific targeting ligand to target an infection site (C); and multifunctional such as theragnostic liposome (D) for diagnosis and treatment. [176]*

Cationic porphyrin 5 -

 5 (1-dodecanoylpyridinium-4-yl)-10,15,20-triphenylporphyrin (TDPyP), incorporated on a poly-cationic LIP named as N-[1-(2,3-dioleoyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTAP) similarly showed inactivation of MRSA.[181]

Figure 1 . 22 .

 122 Figure 1.22. Covalent formulations with aPDT active for PSs.

Figure 1 . 23 .

 123 Figure 1.23. The meso-N-heterocycle BODIPY dyes investigated in Chapter 3.

  Introduction of charge to enhance the aPDT potential Ar = N-heterocycle as meso substituent on BODIPY dye X = Functionality to introduce charge on N-heterocycle functionalized carboxylate derivatives to have a hyper-soluble derivative with tri-cationic species of the molecular scaffold of the BODIPY.

Figure 1 . 24 .

 124 Figure 1.24. B-C derivatives of BODIPY dyes investigated in Chapter 3.

Figure 1 .

 1 Figure 1.24. a) Heavy-atom (iodinated-) derivative b) TMS derivative and c) dimer of BODIPY dyes investigated in Chapter 3.

and chlorins and their application in aPDT 2 . 1 .

 21 photodynamic therapy AlPc: (phthalocyaninato)aluminium(II) BODIPY: Borondipyrromethene difluoroboradiazaindacene BOP: Bleeding on probing CMC: Carboxymethyl cellulose CS: Chitosan CPAT: Chemiluminescent photodynamic antimicrobial therapy ClAlPc: Chloro-aluminium phthalocyanine. CQDs: Carbon quantum dots Ce6: Chlorin e6 CMC: carboxymethyl cellulose CDs: Cyclodextrins DDS: Drug-delivery systems DOTAP: N-[1-(2,3-dioleoyloxy)propyl].-N,N,N-trimethylammonium chloride DPBF: 1,3-Diphenylisobenzofuran EDTA: Ethylenediamine-tetraacetic acid ESBL: Extended-spectrum ß-lactamases EPS: Extracellular polymeric substance ECM: Extracellular matrix EPS: Extracellular polymeric substances EDC.HCl: 1-ethyl-(3-3dimethylaminopropyl) carbodiimide hydrochloride

Figure 2 . 1 .

 21 Figure 2.1. Structure of XF-73 (2.1).

Figure 2 . 2 .

 22 Figure 2.2. Tricationic 2,7,12-tris(trimethyl-p-tolyl)-17-(p-(methoxymethyl)phenyl)porphycene (NMe3MeO-TBPo) (2.2) and 2,7,12-tris(α-pyridinio-p-tolyl)-17-(p-(methoxymethyl)phenyl).porphycene (Py3MeO-TBPo) (2.3).

orFigure 2 . 3 .

 23 Figure 2.3. Structures of cationic porphyrins meso-tetra-(pyridyl)porphyrin (RuTPyP) (2.4) and Zn(II) complex containing peripheral Ru(II)-bipyridyl (ZnRuTPyP) (2.5).

Scheme 2 . 1 .

 21 Scheme 2.1. The biological degradation process of chlorophyll a (2.6) to synthesis of derivatives pheophorbide a (2.6a), chlorin p-6 (2.6b) and purpurin-18(2.6c)

  Photosensitizer

Figure 2 . 5 .

 25 Figure 2.5. Structure of hyaluronic acid (2.16).

Figure 2 . 6 .

 26 Figure 2.6. Schematic illustration of redox-activatable and oxygen self-enriched nano photosensitizers for enhancement tumor photodynamic therapy (PDT).A) The preparation process of PFH@HSC nanoparticles. B) The mechanism of PFH@HSC nanoparticles for selective and efficient PDT against hypoxic tumor cells.[36]*

  with polyethyleneimine (PEI) (600 Da) in CHCl3 for 1 hour and a color change from purple to green. UV-vis spectroscopy confirmed the formation of derivative 2.18 with characteristic of chlorin p6 absorbance at 665 nm (hypsochromic effect of Q (IV) band) as shown in Figure 2.7.

Figure 2 . 7 .

 27 Figure 2.7. UV-vis spectra of Pp-18 (red) (2.17) and Pp-18-PEI (green) (2.18) in CHCl3 .

Scheme 2 . 7 .

 27 Scheme 2.7. Chemical synthesis of 5-(4-acetamidophenyl)-10,15,20-tris(4-pyridyl)porphyrin (2.22) and further introducing cationic charge via methylation to synthesize 5-(4-aminophenyl)-10,15,20-tris(4-Nmethyl pyridinium)porphyrin triiodide (2.24).

  , sulfo-NHS, pH 6.8-7.2,18h, 25 ο C

Figure 2 . 8 .

 28 Figure 2.8. DSC analysis of conjugate formulations a) chlorin p6-PEI-HA (2.25) b) CP-HA (2.26).

Figure 2 . 9 .

 29 Figure 2.9. SEM analysis of conjugate formulation a) chlorin p6-PEI-HA (2.25) b) CP-HA (2.26).

Figure 2 . 10 .

 210 Figure 2.10. IR spectrum of HA-PS conjugates 2.25 and 2.26 in comparison to HA alone.

Figure 2 . 11 .

 211 Figure 2.11. The UV/visible absorption spectra (in water) of PS 2.18 a) Chlorin p6-HA (2.25) and PS 2.24 b) CP-HA after binding to HA (2.26).

( 2 .

 2 25) and CP-HA (2.26)) displayed similar rheological behavior. Indeed, the viscosities of the three samples decreased with increasing shear rate. These results illustrated the shear-thinning behavior of hyaluronic acid and of the two other derivatives. Moreover, above 10 s -1 , all the samples displayed a Newtonian behavior. Looking closer at the impact of the functionalization of HA on the viscosity in this domain, the less viscous sample is chlorin p6-PEI-HA(2.25). The modification of HA with chlorin p6 seems to allow for macromolecular motions, when compared to HA. In addition, CP-HA(2.26) indicated intermediate values of viscosities in the Newtonian area. Thus, intermacromolecular interactions appear to be promoted by cationic porphyrin groups, as compared to chlorin p6.

Figure 2 . 12 .

 212 Figure 2.12. Viscosity (h) as a function of shear rate for HA, chlorin p6-PEI-HA (2.25), and CP-HA (2.26).

Figure 2 . 13 .

 213 Figure 2.13. Storage modulus (G′) and loss modulus (G″) as functions of angular frequency for CP-HA (2.26).

  activity and for CP-HA(2.26) showed comparatively less inhibition as compared to their unbound PSs against the of S. aureus in light and almost negligible activity for E. coli in neither light nor dark conditions as shown in Figures 2.14 and 2.15. Furthermore, to achieve more precise data about the antimicrobial properties of the formulations in comparison with PSs photoinactivation assays were performed. As the formulation of chlorin p6-HA (5) only a negligible inhibition of the bacterial growth no further evaluations were performed.

Figure 2 . 14 .

 214 Figure 2.14. Anti-microbial evaluation of the 5-(4-aminophenyl)-10,15,20-tris(4-Nmethylpyridinium)porphyrin triiodide (2.24) and chlorin p6-PEI (2.18) as compared with hyaluronic acid conjugate derivatives of CP-HA (2.26) and chlorin p6-PEI-HA (2.25) with S. aureus in dark and in light.

Figure 2 . 15 .

 215 Figure 2.15. Anti-microbial evaluation of the 5-(4-aminophenyl)-10,15,20-tris(4-Nmethylpyridinium)porphyrin triiodide (2.24) and chlorin p6-PEI (2.18) as compared with hyaluronic acid conjugate derivatives of CP-HA (2.26) and chlorin p6-PEI-HA (2.25) with E. coli in dark and in light conditions.

Figure 3 . 2 .

 32 Figure 3.2. Singlet oxygen ( 1 O2) sensitive molecular reporters.

Scheme 3 . 1 .

 31 Scheme 3.1. The singlet oxygen reaction of DPBF (3.5) to produce 3.11 degraded product.

Figure 3 . 3 .

 33 Figure 3.3. Library of BODIPY-anthracene (3.12-3.17) and -pyrene (3.18) dyads and phenyl (3.19) and their relative singlet oxygen quantum yields (Ф∆) in EtOH.

3. 12 : 59 3. 18 : 1 Figure 3 . 4 . 39 3. 24 :Figure 3 . 5 . 84 3. 30 :

 1259181343924358430 Figure 3.4. Library of water-soluble anthracene-(3.20-3.25), pyrene-(3.26) or phenyl-(3.27) BODIPY dyads and their relative singlet oxygen quantum yields (Ф∆) in EtOH.

Figure 3 . 6 .Figure 3 . 7 .Figure 3 . 8 .

 363738 Figure 3.6. Structures of a) di(p-benzi)homoporphyrin derivatives (3.31-3.33) and b) di(mbenzi)homoporphyrin (3.34-3.36) derivatives.These PSs exhibited a strong absorption band between 600-700 nm and act as efficient photosensitizers for the formation of singlet oxygen. UV absorbance degradation assay of DPBF in the presence of di(p/m-benzi)homoporphyrin derivatives(3.31-3.36) and compared to reference PS, H2TPP (degradation plots in Figure3.7).[17] UV-degradation plots and time-dependent degradation curves measured in DCM:MeOH (1:1) are illustrated in Figure 3.8 (di(p-benzi)homoporphyrin (3.31-3.33)) and Figure 3.9 (di(mbenzi)homoporphyrin (3.34-3.36)) respectively. The derived values for relative quantum yields are collated in Table 3.2.

Figure 3 . 9 .

 39 Figure 3.9. Time dependent DPBF degradation via di(p/m-benzi)homoporphyrin derivatives (3.31-3.36) at 415 nm.

  decrease in DPBF UV-absorption and time dependent degradations for BODIPY-TPE (3.37 and 3.38) derivatives is shown in Figures 3.11 and 3.12. Similar to the previous experiment the DPBF degradation assay was measured in DCM:MeOH (1:1)in a concentration of 0.15 mol.L -1 . The relative quantum yields were calculated with reference to H2TPP as shown previously in Figure3.7.[17]

Figure 3 . 10 .Figure 3 . 11 .Figure 3 . 12 .

 310311312 Figure 3.10. Tetra-BODIPY-appended TPE derivatives (BODIPY-TPE (3.37 and 3.38)) used for DPBF degradation assay for singlet oxygen production.

Figure 3 . 13 . 41 UV

 31341 Figure 3.13. A series of brominated porphyrins (3.39-3.41).

Figure 3 .

 3 Figure 3.14. a) UV-Vis spectra of DPBF degradation and (b) DPBF consumption via MB at 415 nm.

Figure 3 . 15 .

 315 Figure 3.15. DPBF consumption measured at 415 nm over time for brominated porphyrin derivatives (3.39-3.41) derivatives.

Figure 3 . 16 .

 316 Figure 3.16. UV-vis DPBF consumption measured at 415 nm over time for brominated derivatives of porphyrins (3.39-3.41).

amount of 1

 1 photoactivity of such molecules.Constant efforts are put in by scientists to synthesize PSs active for PDT by functionalizing the macrocycles of known PSs to develop the best candidate for therapeutics. This functionalization enhances the photoactivity of the molecules and thus they can be pre-evaluated with DPBF degradation assays and further subjected to photophysical evaluations or for biological assays. Thus, such probes have been a vital tool for assessment of the applicability of the PSs based on tetrapyrroles or dipyrromethene dyes as synthesized in the lab in pre-clinical studies.

Figure 4 . 1 .

 41 Figure 4.1. BODIPY dye core and its IUPAC numbering system.

Scheme 4 . 1 .

 41 Scheme 4.1. Synthetic route to meso-alkyl-substituted BODIPY (4.5) dyes using acyl chloride(4.3)

an oxidizing agent 2 , 3 -

 23 dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) yielding dipyrromethene or dipyrrin which is stable. Without further purification, under basic conditions, boron trifluoride (as BF3•Et2O) is added to the crude dipyrrin (4.9), which results in the formation of highly fluorescent, red-to-orange colored BODIPY (4.10) dye in 10-30% from starting materials. This reaction can be performed in an organic solvent (e.g., dry CH2Cl2); for an unsubstituted pyrrole precursor (R1-R3 = H), the pyrrole itself is used as reaction solvent, to prevent porphyrin or oligomer formation by significant excess of this reagent. Products which can be formed without the use of chlorinated solvents are far more environmentally friendly.

Figure 4 . 2 .

 42 Figure 4.2. Cationic BODIPY dyes derivatives (4.11 and 4.12) active for aPDT.

Figure 4 . 3 (

 43 BODIPY dye derivatives 4.13-4.22)

Figure 4 . 3 .

 43 Figure 4.3. BODIPY dye derivatives with a) mono-and brominated BODIPYs bearing electron-donating substituents and BODIPYS and meso-acetoxymethyl moiety (4.13-4.22) and b) substitution at 2,6position with halogens such as bromine (4.23) and iodine (4.24) atoms.

Figure 4 . 4 ,

 44 showed an enhancement in the singlet oxygen

Figure 4 . 4 .

 44 Figure 4.4. Conjugation of BODIPY (4.25) dyes with AuNRs (4.26).

35 and 4. 40 - 4 . 41 )Figure 4 . 5 .

 4044145 Figure 4.5. Conjugation of BODIPY dyes: a) amine (4.27-4.33) b) thio (4.34-4.41) carbohydrates.

Figure 4 . 6 .

 46 Figure 4.6. Side product, tripyrrin 4.59a obtained whilst synthesis of BODIPY 4.59.

Scheme 4 . 4 .

 44 Scheme 4.4. Synthesis of charged water-soluble meso-methyl-BODIPYs 4.46a, 4.54a, 4.48a and 4.56a.

BODIPYs 4 .Scheme 4 . 5 .

 445 Scheme 4.5. Synthesis of charged water-soluble meso-methyl benzoate-BODIPYs 4.46b, 4.54b, 4.48b and 4.56b.

Scheme 4 . 6 .

 46 Scheme 4.6. Synthesis of charged water-soluble meso-butylphenyl etherate-BODIPYs 4.46b, 4.54b

Scheme 4 . 7 .

 47 Scheme 4.7. Synthesis of boron-ethynyl-substituted carboxylate derivatives (4.46d and 4.50a).

Scheme 4 . 8 . 4 . 5 . 1 . 4 .

 484514 Scheme 4.8. Synthesis of 2-functionalized iodinated derivatives (4.46e, 4.49a, 4.51a and 4.53a)

Scheme 4 . 8 .

 48 Scheme 4.8. Synthesis of extended π-conjugated BODIPY (4.66).

  properties. All BODIPYs were characterized by1 H-and 13 C-NMR, HRMS and by X-ray crystallography. Analysis of the NMR spectra shows the anticipated 2-fold symmetry of the BODIPY. Single signals for the 2,6-hydrogens, the 1,7-and 3,5-dimethyl groups for 2,4-dimethyl pyrrole derived BODIPYs (4.46-4.53) and 2,6-diethyl, the 1,7-and 3,5dimethyl groups for 3-ethyl-2,4-dimethyl pyrrole derived BODIPY (4.54-4.60) were observed in the 1 H-NMR spectra and 13 C signals for only half of the BODIPY core carbons in 13 C-NMR specifying a plane of symmetry extending through the boron and C-8. meso-Aryl substituted groups show characteristic 1 H proton resonances and carbon signals corresponding to aryl group carbons. Again, NMR resonances shifts for the BODIPY core were relatively same for different meso-aryl functionalization with water-soluble functionalization on the N-heteroatom of the meso-N-heterocycle for groups such as methyl (4.46a, 4.54a, 4.48a and 4.56a), carboxylate (4.46b, 4.54b, 4.48b and 4.56b) and etherate (4.46c and 4.54c) or for boron-ethynyl-substituted BODIPY derivatives (4.46d and 4.50a). The observed up-field shift in 1 H NMR for iodinated derivatives (4.46e, 4.49a, 4.51a and 4.53a) of the methyl groups in positions 1 and 7 (at approximately 1.6 ppm) compared with the 3,5-dimethyls (at approximately 2.5 ppm) is likely caused by shielding incurred by the meso-aryl groups. The lack of signal of the 2-hydrogen (at ~ 6.0 ppm) in the 1 H-NMR spectra indicated monoiodination. Similar resonance shifts are observed TMS functionalized derivatives 4.46f and 4.51b and TMS-deprotected derivative 4.46g.

  state and lodged with the Cambridge Crystallographic Data Centre (CCDC), a repository which catalogues this information. Among the most popular sub-classes of studied materials are the variously substituted meso-phenyl (1031 examples) 1,3,5,7tetramethyl (776 examples) and aza-BODIPYs (i.e. 4,4-difluoro-3a,4a,8-triaza-4-boras-indacene, 80 examples. Examples of crystal structures of (meso-Ar) BODIPY compounds with Ar being an N-heterocycle include Ar = pyridine, quinoline, pyrimidine,

Figure 4 . 7 . 59 Figure 4 . 8 .Figure 4 . 9 .Figure 31 .

 4759484931 Figure 4.7. View of the molecular structures of the BODIPYs 4.47, 4.49, 4.50 and 4.52 in the crystals. Thermal ellipsoids are plotted at 50%, H-atoms are represented as spheres of fixed radius.

55 Figure 4 . 10 .

 55410 Figure 4.10. Intermolecular C-H⋯N interactions engaged by BODIPYs 4.47(a) (C24⋯N21 3.42 Å, C22⋯N23 3.46 Å) and 4.55(b) (C26⋯N25 3.43 Å). Methyl and ethyl groups on the BODIPY core are omitted.

Figure 4 .

 4 11, for BODIPY 4.50.

Figure 4 . 11 .

 411 Figure 4.11. C-H⋯N and C-H⋯F "weak" interactions engaged by BODIPY 4.50, C28⋯F17 3.347 Å; C26⋯F18 3.267 Å; N20⋯C24 3.621 Å.

Figure 4 . 12 .Figure 4 . 13 .

 412413 Figure 4.12. View of the molecular structures of the BODIPYs 4.48a and 4.54c in the crystals. Thermal ellipsoids are plotted at 50%, H-atoms are represented as spheres of fixed radius.

Figure 4 . 14 .

 414 Figure 4.14. The four inequivalent molecules in the structure of BODIPY 4.48b due to disorder present in solution

11 ( 4 . 12 ( 4 .

 114124 The B-F distances, with a mean value of 1.393 Å, are additionally affected by the environment of the fluorine atoms; there is a clear indicator that the values for these cationic compounds are statistically longer when compared to the distribution for all BODIPY compounds in the literature (1.387(17) Å) with p = 0.03 for a one-tailed t-test.Each of these molecules engages in significant 'weak' interactions, a catch-all term for hydrogen bonding, π-stacking and other associative interactions generally weaker than a coordinate bond. Interactions with solvate (e.g. 2.720(9) Å O⋯F and 161(18) ° O-H⋯F H-bond from water in compound 11) are comparatively strong; charge-assisted interactions between pyridinium C-H and Br -counter-anion (e.g. C⋯Br, Figure4.48a)), and between protonated carboxylate and counter-anion (e.g., Figure4.54b and 4.56b)) are observed.

  hence the optical window of such compounds. The three compounds herein are intermediates in the synthesis of an exemplar dyad -with ethynyl units and with the butadiyne-bridged dimer structure. Molecular units of the two precursor BODIPYs 4.46f and 4.46g are shown in Figure 4.15; in each case, no additional solvent was included within the unit cell.

Figure 4 . 15 .

 415 Figure 4.15. View of the molecular structures of the BODIPYs 46f and 4.46g. Thermal ellipsoids are plotted at 50%, H-atoms are represented as spheres of fixed radius.

Figure 4 .Figure 4 .

 44 Figure 4.16. a) C-H⋯N interactions within the crystal structure of BODIPY 4.66; b) these interactions form three, three-dimensional hydrogen-mediated networks in red, green, and blue.

Figure 4 . 17 .

 417 Figure 4.17. (a,b); Molecular structures of the dominant components of 4.52a; (c,d) a side-on view of the molecules in 4.52a (a and b) showing disorder of the methyl and ethyl groups on the pyrrole backbone in this crystal structure. H atoms are omitted, with non-H atom ellipsoids at 50%.

Figure 4 . 18 .

 418 Figure 4.18. UV-vis absorption (nm) for BODIPY derivative 4.47 (1,3,5,7-tetramethyl derivative) and 4.55 (2,6-diethyl-1,3,5,7-tetramethyl derivative) in DCM.

of 2 -

 2 position of BODIPYs 4.49a and 4.51a, caused a bathochromic shift in absorption as expected, into the orange/red spectral region as compared to their parent BODIPY (4.49 and 4.51) An example of this change is illustrated in Figure 4.19 where the UV-absorption maximum for derivative 4.51a is red-shifted by 31 nm as

Figure 4 . 19 .

 419 Figure 4.19. UV-vis absorption (nm) for BODIPY derivative 4.51 (Parent BODIPY) and 4.51a (Iodinated derivative) in DCM.

Figure 4 . 20 .

 420 Figure 4.20. UV-vis absorption (nm) for BODIPY derivative 4.46g (Parent BODIPY) and 4.66 (conjugated derivative) in DCM.

Figure 4 .Table 4 . 8 . 53 ( 1 , 3 , 5 , 7 -

 448531357 Figure 4.21. a) UV-Vis spectra of DPBF degradation of Rose Bengal (RB) (reference) and (b) DPBF consumption via RB measured at 415 nm in DCM/MeOH.

Figure 4 . 22 .

 422 Figure 4.22. UV-vis DPBF consumption measured at 415 nm over time for meso-N-heterocycle substituted BODIPYs 4.46-4.53 (1,3,5,7-tetramethyl substituted) in DCM/MeOH.

Figure 4 . 23 .Figure 4 . 24 .

 423424 Figure 4.23. UV-vis DPBF consumption measured at 415 nm over time for meso-N-heterocycle substituted BODIPYs 4.54-4.60 (2,6-diethyl -1,3,5,7-tetramethyl substituted) in DCM/MeOH.

Figure 4 . 25 .

 425 Figure 4.25. UV-vis DPBF consumption measured at 415 nm over time for charged water-soluble etherate derivatives (4.46c and 4.54c) in DCM/MeOH.

Figure 4 . 26 .Figure 4 . 27 .

 426427 Figure 4.26. UV-vis DPBF consumption measured at 415 nm over time boron-ethynyl functionalized precursor scaffolds (4.46d and 4.50a) in DCM/MeOH.

4. 5 . 5 .-vitro studies 4 . 5 . 5 . 1 .

 554551 InInhibition assays (MIC/MBC) The inhibition assays of the compounds were performed with help of Ms. Charlotte Vernisse. Photo-induced antibacterial activity of synthesized BODIPY dyes were tested against two model organisms, the Gram-negative P. aeruginosa and the Gram-positive S. aureus, the minimal inhibitory concentration (MIC) and the minimal bactericidal concentration (PD-MBC) under light irradiation (25 J/cm²). The results (Tables 4.10 and 4.11) clearly indicate that different meso substituents of BODIPY dyes and difference in 2-6-subtitution impact the efficiency for aPDT. BODIPYs 4.47, 4.53 and 4.55 display an efficiency at <50 µM (MIC/MBC) for inhibition of S. aureus. Iodinated derivatives (4.46e, 4.49a and 4.53a) as also display efficient inhibition at <100µM (MIC/MBC) of S. aureus.

4. 5 . 5 . 2 .

 552 Biofilm inhibition and eradication Inhibition of the biofilm for BODIPYs 4.56b and 4.54c was tested against S. aureus and both the BODIPYs showed a decrease of biofilm growth under light illumination as shown in Figure 4.28 and 4.29, respectively.

Figure 4 . 28 .

 428 Figure 4.28. Percentage inhibition of the biofilm growth by BODIPY 4.56b under dark and light conditions

Figure 4 . 29 .

 429 Figure 4.29. Percentage inhibition of the biofilm growth by BODIPY 4.54c under dark and light conditions

Figure 4 . 30 . 4 -

 4304 Figure 4.30. Schematic illustration to synthesize oxidized CNCs

  using a Quanta FEG 450 ESEM with an accelerating voltage of 10-15 kV. Conjugates were fixed onto carbon-taped metal pins. Dynamic light scattering (DLS) was used for the measurement of average hydrodynamic diameters and polydispersity indexes (PDIs; Malvern Zetasizer Nano-ZS, Malvern Instruments, UK). Each sample was analyzed in triplicate at 20 °C at a scattering angle of 173°. Pure water was used as a reference dispersing medium. The ζ-potential data were collected through electrophoretic light scattering at 20 °C, 150 V, in triplicate for each sample (Malvern Zetasizer Nano-ZS, Malvern Instruments, UK). The instrument was calibrated with a Malvern -68 mV standard before each analysis cycle. Differential scanning calorimetry (DSC) analysis was carried out on a Model 910 Differential Scanning Calorimeter. Dynamic rheology properties were obtained using a rotational rheometer (Mars III, Thermo Scientific, RheoWin software), with a 35-mm plate-plate geometry configuration and a gap of 1.000 mm. The behavior of the different samples was determined by shear rate sweep tests at 25 °C. In addition, the rheological properties were measured in strain-controlled oscillatory tests. A strain amplitude of γ0 = 0.1 [-] was selected to ensure a linear regime of oscillatory deformation. The conjugate was placed between the plates at 25 °C, and the measurements were carried out within the angular frequency range of 0.1-100 rad/s.

  , 1H, 5-H, 10-H, 20-H); 7.86 (dd, J = 17.0, 11.4 Hz, 1H, 31-CH); 6.28 (dd, J = 17.0, 1.0 Hz, 1H, trans-32-CH2); 6.19 (dd, J = 11.4, 1.0 Hz, 1H, cis-32-CH2); 5.15 (m, 1H, 17-H);

[ 3 ]

 3 M.p. = >300°C; UV-visible (MeOH), λmax (ε [x10 3 M -1 cm -1 ]): 428 (5.05), 521 (4.05), 564 (3.88), 593 (3.84), 656 nm (3.47). 1 H NMR (500 MHz, DMSO d6): δ (ppm) = 9.49 (d, J = 6.5 Hz, 6H, Ho-py), 9.11 -8.80 (m, 8H, Hpyrr), 9.00 (d, J = 6.5 Hz, 6H, Hm-py), 7.94 (d, J = 8.5 Hz, 2H, Ho-phe), 7.15 (d, J = 8.5 Hz, 2H, Hm-phe), 4.73 (s, 9H, N+ -CH3), -2.96 (s, 2H, NHint). HRMS (ESI+): m/z calcd for C44H37I3N8 [M] 3+ 225.77, found 225.74. conjugates: Thermogram shows a loss of water (endothermic) from around 80 °C, then an exotherm at about 240 °C for this hyaluronic acid conjugate. SEM characterization: Representative SEM images (Figure S2) of the dried conjugate samples (with PSs) shows homogenous dispersion of the conjugate samples and crosslinking pattern with an average size of 105 ± 103 nm for 2.25. IR spectrum: 3360, 3325, 2930, 2947, 1642, 1574, 1596 and 1340 cm -1 .

  conjugates: Thermogram shows a loss of water (endothermic) from around 80 °C, then an exotherm at about 240 °C for hyaluronic acid conjugate. SEM characterization: Representative SEM images (Figure S2) of the dried conjugate samples (with PSs) shows homogenous dispersion of the conjugate samples and crosslinking pattern with an average size of 215 ± 105 nm for this conjugate. IR spectra: 3360, 3325, 2930, 1642 and 1340 cm -1 . DLS analyses and zeta potential analyses of conjugates: Hydrodynamic diameter of this conjugate is 435.1 ± 35.1 nm and zeta potential is -29.3.

[ 4 ] 1 , 3 , 5 , 7 -

 41357 M.p. = 235 -240 °C; Rf = 0.52 (DCM/Hex: 90/10); 1 H NMR (400 MHz, CDCl3): δ = 8.78 ppm (dd, J = 4.3, 1.6 Hz, 2H, Ar-H), 7.30 (dd, J = 4.3, 1.6 Hz, 2H, Ar-H), 6.00 (s, 2H, 2-H, 6-H), 2.55 (s, 6H, 3-CH3 and 5-CH3), 1.40 (s, 6H, 1-CH3 and 7-CH3). 19 F NMR (377 MHz, CDCl3): δ = -145.90 --146.39 ppm (m); 11 B NMR (128 MHz, CDCl3): δ = 0.70 ppm (t, J = 32.5 Hz); IR (neat): ν = 2963, 1536, 1436, 1360, 1138, 1049, 962 cm -1 . HRMS (ESI): m/z calcd for C18H18BF2N3 [M + H] + : 326.163789, 326.163915 found. Tetramethyl-8-(5-pyrimidinyl)-4-bora-3a,4a-diaza-s-indacene (4.47) BODIPY 4.47

(ε [x10 3 M 1 , 3 , 5 , 7 -

 31357 -1 cm -1 ]) = 319(5.54), 369(6.18), 481(24.92), 512 (105.42) nm. HRMS (APCI): m/z calcd for C17H17BF2N4 [M + H] + : 327.159021, 327.158854 found. Tetramethyl-8-(4-quinolinium-yl)-4-bora-3a,4a-diaza-s-indacene (4.48) BODIPY 4.48 In accordance with general procedure B as described above using 2,4-dimethylpyrrole (2.0 mL, 19.4 mmol) and 4-quinoline carboxaldehyde (1.41 g, 9 mmol) and final product was obtained and purified by column chromatography (silica gel; DCM/Hex, 7:3, v/v) and removal of solvents followed by recrystallization (DCM/Hex) to yield green solid (20% yield). M.p. = 252 -257 °C ; Rf = 0.48 (DCM/Hex: 90/10); 1 H NMR (400 MHz, CDCl3) δ 9.03 (d, J = 4.3 Hz, 1H, Ar-H), 8.19 (d, J = 8.5 Hz, 1H, Ar-H), 7.84 (d, J = 8.3 Hz, 1H, Ar-H), 7.78 (ddd, J = 8.3, 7.0, 1.2 Hz, 1H, Ar-H), 7.56 -7.51 (m, 1H, Ar-H), 7.39 (d, J = 4.3 Hz, 1H, Ar-H), 5.97 (s, 2H, 2-H, 6-H), 2.59 (s, 6H, 3-CH3, 5-CH3), 1.09 (s, 6H, 1-CH3, 7-CH3).

970 cm - 1 .

 1 UV-Vis (CH2Cl2) λmax (ε [x10 3 M -1 cm -1 ]) = 314 (3.14), 369 (4.36), 486 (12.00), 519 (46.15) nm. HRMS (APCI): m/z calcd for C17H19BF2N4 [M + H] + : 329.174672, 329.174808 found. 1,3,5,7-Tetramethyl-8-phenyl-4-bora-3a,4a-diaza-s-indacene (4.53) BODIPY 4.53

  F NMR (377 MHz, CDCl3) δ -144.88--146.38. 11 B NMR (128 MHz, CDCl3) δ 0.75 (t, J = 32.5 Hz). IR (neat): 3110, 2961, 2925, 1596, 1539, 1397, 1182, 969 cm -1 . UV-Vis (CH2Cl2) λmax (ε [x10 3 M -1 cm -1 ]) = 337 (3.15), 394 (9.12), 513 (19.64), 547 (54.73) nm. HRMS (APCI): m/z calcd for C20H24BF2N3S [M + H] + : 388.182851, 388.183008 found. The formation of the BODIPY 4.59 resulted in a side-product (Z,Z)-2,5-bis(2thiazolylmethanylidene)-3-ethyl-4-methylpyrrole (4.59a).

90/ 10 )

 10 ; 1 H NMR (400 MHz, CDCl3) δ 8.81 (d, J = 0.6 Hz, 2H, Ar-H), 7.30 (d, J = 4.7 Hz, 2H, Ar-H), 6.06 (s, 1H, 6-H), 3.30 (s, 3H, 2-CH3), 2.61 (d, J = 23.3 Hz, 6H, 3-CH3, 5-CH3), 1.45 (d, J = 21.2 Hz, 6H, 1-CH3, 7-CH3).

1 Equation 1 ;

 11 photosensitizer and reference molecule (H2TPP, RB or MB), respectively.

BODIPY 4 .BODIPY 4 . 66 :

 4466 56b: Squeeze was used to account for disordered solvent (See: Spek, A. L., ActaCryst.(2015). C71, 9-18 doi:10.1107/S2053229614024929). The four-equivalent solvent-containing cavities sum to 17% of the total volume of the crystal (625 Å 3 ), slightly smaller than that expected for four ethyl acetate molecules at room temp. (650 Å 3 ). BODIPY 4.54c: generally poor diffraction at high angles, and unresolved dual pseudomerohedral twinning issues. Large Fourier ripples from Br. BODIPY 4.49a: Solution has residual Q-peaks from Fourier ripples from the I atom (Balert, explicable). No special refinement. BODIPY 4.46f: No special refinement necessary. BODIPY 4.46g: No special refinement necessary. No special refinement necessary. BODIPY 4.52a: Disorder of the methyl and ethyl groups was fixed with PARTs 1,2,3 and SUMP for C50-57. SIMU was used to fix thermal parameters in the disordered fragments, and SADI was used to fix equivalent bonds between disordered atoms.

chapter 3 ,

 3 photodynamic therapy (aPDT)

Table 1 . 1 .

 11 Commonly studied PSs and their photodynamic properties

	Class	Example	Charge	Excitation	Sample	Concentration	Reduction	Ref.
				maximum	(bacteria)	of PS	of bacterial
							cultures
	Pheno	Methylene blue	Cationic	632nm	Dental plaques	25 µg/ml	8%	49
	-thiazinium				sample
		Toluidine blue	Cationic	410nm	S. mutans	100 mg/l	2-5 log10	50,
					E. coli	35 µM	0.08 log10
		Rose Bengal	Cationic	532nm	E. faecalis	10 µM	4 log10	52
					P. aeruginosa		3 log10
		Dimethyl methylene	Cationic	635-652nm	A. baumannii	200 µM	2 log10	53
		blue
		New methylene blue	Cationic	635-652nm	A. baumannii	800 µM	3.2 log10	54
		Curcumin	Neutral	547nm	S. mutans	0.75 to 5 g/l	≥ 3 log10	55,
	Natural PSs				L. acidophilus
		Hypericin	Neutral	593nm	S. aureus	100 nM	4-5 log10	57,
					E. coli	1 µg/ml	≤ 0.2 log10
		Flavin derivatives	Cationic	450nm	MRSA	50 µM	5.1 log10	59
					Enterohemorrhagic	50 µM	6.5 log10
					E. coli
		Chlorophyllin	Neutral	400nm	Bacillus cereus	150 µM	2.4 log10	60
					Listeria monocytogenes
		Phenalenone	Neutral	363 nm	S. Aureus	5 µM	9 log10	61
					E. faecalis	3 µM	3 log10
	Tetra-	Porphyrin derivatives	Cationic	446nm	S. aureus	10 µM	1-2 log10	62-64
	pyrrole				P. aeruginosa	225 µM	4 log10
	structures				E. faecalis	100 µM	No effect
		Phthalocyanine	Neutral	670nm	A. hidrophila	2 mM	≤ 0.5 log10	65
		derivatives (Pc)
		Zn(Pc)	Cationic	690nm	S. aureus	64 ng/ml	5-6 log10	66-67
					P. aeruginosa	26 µg/ml	5-6 log10
		Chlorin derivatives	Neutral	660nm	S. aureus	10 µg/ml	5 log10	68
					E. coli	5 µg/ml	0.75 log10
		Chlorin derivatives	Cationic	532nm	E. coli	5 µg/ml	0.77 log10	69

Table 1 . 2 .

 12 Drug delivery systems based on biopolymers with characteristics used with a focus on aPDT improvement.

	System	Approach	PS/λ(n	Microorganism(s)
			m)

1.3.2.2. Synthetic-polymer derived hydrogels with PSs

	In aPDT, polymeric hydrogels have been used to encapsulate PSs via loading or are
	built from polymers known to have inherent photoactivity against microbes. Several PSs
	(RB, porphyrins and derivatives, curcumin, among others) have been encapsulated in
	biocompatible polystyrene (1.19), polyethylene glycol (PEG) (1.20), polyester (1.21), or
	polyacrylamide (PAA) (1.22) based polymers (Figure. 1.16) and used in aPDT.
	C C H	H	O	O	H
	H	H n		n
	1.19			1.20
	O			H 2 C	H C
	O R	n		O NH 2	n
	1.21			1.22
	Figure 1.16. Examples of synthetic polymers (polystyrene (1.19), PEG (1.20), polyester (1.21) and PAA
	(1.22)) used for hydrogel formation with PSs.

1.3.2.3.2. Chitosan

				This was shown by incubating the
	bacteria	suspension	of	Gram-positive, S.	aureus and	Gram-negative, E.
	coli and Pseudomonas aeruginosa with both photoactive cellulosic fabrics in the dark
	and in light with different light intensities and time-intervals. Although the Gram-negative
	bacteria were less susceptible to this aPDT regimen than the Gram-positive bacterium, Figure 1.19. Cellulose derivatization to form antibacterial surfaces with PSs. [150]
	the cellulose based ZnTMAP 4+ photoactive material showed remarkable efficacy
	against P. aeruginosa, highlighting the role of core modification in biocidal effects. Sodium alginate (SA), pectin (PC), and carboxymethyl cellulose (CMC) have also been
	The Sol laboratory in Limoges, where work presented in this manuscript was performed, used for preparing films containing chlorin p6 or MB. The photodynamic efficacy of Cp6-
	also obtained a novel antimicrobial platform by grafting PS, 5-(4-propargyloxyphenyl)-SA-PC-CMC films is higher for MRSA as compared with MB-SA-PC-CMC films due to
	10,15,20-tritolylporphyrin(Zn 2+ ) (1.28) on cotton fabric derived cellulose via azidation insignificant binding to the film components. [151]
	(1.23a) followed by "Click-Chemistry" with acetylenic group of the porphyrin in Figure
	1.19. This material displayed an antimicrobial action against E. coli and S. aureus,
	making this platform a potential photo-bactericidal formulation (1.29) applicable for
	industrial as well as for household utilizations. [150] Chitosan (CS) (1.24) is a naturally occurring polycationic polymeric natural structural
	element of fungal cells walls and crustacean exoskeletons, usually obtained by chitin
	acid hydrolysis. It is composed of N-acetyl-D-glucosamine and 𝛽-1,4-linked D-

HA)-PS conjugates (chlorin p6-PEI-HA (2.25) and CP- HA (2.26))

	+ Hyaluronic acid (These two PSs, 2.18 and 2.24, were further covalently grafted on hyaluronic acid (HA) + Propionic acid 4 3 2.3.3.
	140 ο C, 1.5h as shown in Scheme 3 via an amide link yielding conjugate chlorin p6-PEI-HA (2.25)
	and CP-HA (2.26). The conjugates with HA were synthesized in aqueous media thus
	2.19 carboxylic acid activation of HA (2.16) was achieved by EDC/NHS as shown in Scheme 2.20 2.21
	2.8.
	2.22
	a) Excess Iodomethane (2.23), anhydrous DMF, 140 ο C, 3h	b) 25% TFA/H2O, 100 ο C, 5h
		.3I -
		2.24

Scheme 2.8. PSs 2.18 and 2.24 covalently grafted on hyaluronic acid (HA) (2.16) via an amide link yielding HA-PS conjugates 2.25 and 2.26.

Culture media Gentamycin 2.18 2.24 Culture media Gentamycin 2.18 2.24 2.16 GM-HA 2.26 2.25 2.16 GM-HA 2.26 2.25

  The photodynamic activity of the photosensitizer (2.24) conjugated with HA (2.26) was tested in vitro against S. aureus, as a model of Gram-positive bacteria. The experimental results are reported in Table2.1. The microplate was incubated at 37°C for 5h with white

	2.4.2.	Photoinactivation of bacterial cells
	Culture media LED light (total fluence: 25J/cm²) or in the dark. Then, 500 µL of 2-fold concentrated TS Gentamycin 2.16 GM-HA
	broth and glucose (25mM) were added in each well and the microplate was incubated
	at 37°C for 24h.
		2.18	2.24	2.25	2.26
		Culture media	Gentamycin	2.16	GM-HA
		2.18	2.24	2.25	2.26

Table 2 . 1 .

 21 MIC (µM) evaluation of CP-HA (2.26) as compared with the individual PS(2.24)

	Bacterial	PS/PS-HA	MIC	MIC
	strains	conjugates
			Dark	Light
	S. aureus	2.24	200 µM	100 µM
		2.26	> 700 µM	700 µM
	E. coli	2.24	> 200 µM	200 µM
		2.26	> 700 µM	700 µM

Table 3 .2. Relative

 3

singlet oxygen production of for di(p/m-benzi)homoporphyrins (

3.31-3.36) in

DCM/MeOH (1:1) using H2TPP, ΦΔ = 0.62 as a reference.

Di(p/m-benzi)homoporphyrin derivatives Singlet oxygen quantum yield

		(ΦΔ)
	3.31	0.11
	3.32	0.23
	3.33	0.05
	3.34	0.11
	3.35	0.14
	3.36	0.07

Table 3 . 3

 33

. Relative singlet oxygen production of for BODIPY-TPE (3.37 and 3.38) in DCM/MeOH (1:1) using H2TPP, ΦΔ = 0.62 as a reference.

Table 6 .

 6 Relative singlet oxygen production of for brominated derivatives DCM/MeOH (1:1) using MB, ΦΔ =0.50 as a reference.

Table 4 . 1 .

 41 Photodynamic minimal inhibitory concentrations (MIC) of BODIPYs 4.22 and 4.12 for S.

		xylosus and E. coli irradiated at 20.7 W/cm 2
	BODIPY	Gram-bacteria MIC (µM)
		E. coli
	4.11	1.25 ± 0.57
	4.12	6.60 ± 2.88
		S. xylosus
	4.11	0.31 ± 0.13
	4.12	0.83 ± 0.36

Table 4 . 2 .

 42 Percentage yields of new meso-aryl-BODIPY derivatives (4.46-4.60).

	3. A library of

Scheme 4.3. Synthesis of library of meso-aryl-BODIPYs. aldehyde, thus introducing partial positive charge on the carbonyl carbon and hence promoting nucleophilic attack by pyrrole. TFA facilitates this process to form dipyrromethane (4.44) derivative, which is relatively unstable. After 12 h of stirring at BODIPY 4.59, resulted from a rearrangement mechanism is such reaction conditions.

Table 4 . 3 .

 43 Percentage yields of water-soluble BODIPY derivatives.

	BODIPY	Ar	% Yield	BODIPY	Ar	% Yield
	4.46a		81	4.54b		75
	4.54a		73	4.48b		63
	4.48a		85	4.56b		68
	4.56a		75	4.46c		70
	4.46b		82	4.54c		72

Table 4 . 4 .

 44 . Compounds reported herein are shown in Figures 4.7-4.9 and Table8, with selected structural data listed in Table4.4. The N-heterocyclic group at the meso-position in each example is one of 4-pyridyl, 2-quinolinyl, 4-Compound formulae for the crystal structure determinations of neutral N-heterocyclic

	quinolinyl, 1-methyl-2-imidazolyl, 2-thiazolyl, 5-pyrimidinyl or 9-acridinyl, and the
	BODIPY substitution pattern is 1,3,5,7-tetramethyl (denoted Me4) (4.47, 4.49, 4.50 and
	4.52) , 2,6-diethyl-1,3,5,7-tetramethyl (Et2Me4) (4.55, 4.56, 4.57, 4.59 and 4.60) or 2-
	Iodo-1,3,5,7-tetramethyl (Me4I) (4.49a). In each case, the crystal structure acts as
	verification of the structural assignment, in agreement with NMR. Beyond using these
	structural determinations for the assignment of structure, we can investigate the
	conformational nature of the individual BODIPY components using symmetry
	coordinates. This methodology is intended to provide a single, simple value for

49 BODIPY 4.50 BODIPY 4.52

Table 4 . 5 .

 45 Plane-to-plane angles of the meso-substituent and BODIPY system and C-C bridgehead distances* in each of the cationic BODIPY compounds and their neutral counterparts.

	BODIPY	Plane-Plane	P/P for	C-C bridge	C-C in	B-F (Å)
		angle (°)	neutral comparison	head (Å)	neutral (Å)
			compound (°)
	BODIPY 4.48a	84.96(9)	79.102(19)	1.496(10)	1.4899(15)	1.370(11)
						1.410(11)
	BODIPY 4.54c	79.51(13)	81.328	1.499(7)	1.483(5)	1.381(7)
					(10.1039/C4CP03	1.395(8)
					343D)
	BODIPY 4.54b	85.656 (44)	79.102(19)	1.494(3)	1.4899(15)	1.395(3)
						1.395(2)
	BODIPY 4.56b	87.946(81)	81.328	1.496(6)	1.483(5)	1.386(8)
					(10.1039/C4CP03	1.397(7)
					343D)
	BODIPY 4.48b (C1)	73.14(7)	81.328	1.482(4)	1.483(5)	1.380(4)
						1.411(4)
	BODIPY 4.48b (C35)	79.99(6)		1.483(3)		1.395(3)
						1.398(3)
	BODIPY 4.48b (C69)	75.71(5)		1.483(4)		1.390(3)
						1.405(4)
	BODIPY 4.48b (C103) 75.91(5)		1.490(4)		1.391(3)
						1.402(3)

* The bond involves two inverted carbon atoms, whose hybrids point away from each other.

Table 4

 4 and 547 nm for 3-ethyl 2,4-dimethyl derived meso-aryl BODIPYs (4.54-4.60) (Table4.6). The main point of differentiation of the BODIPY dyes is with different substitution patterns on the pyrrole units. The 1,3,5,7-tetramethyl substituted BODIPY

	and 2,6-diethyl-1,3,5,7-tetramethyl substituted BODIPY derivative are differentiated by
	a red shift of 20-25 nm on introduction of the ethyl groups. For comparison, 5-
	pyrimidylBODIPY derivatives (4.47) (1,3,5,7-tetramethyl) and (4.55) (2,6-diethyl-1,3,5,7-

.6. There was little to no solvent effect observed in the absorption maxima wavelengths. The newly synthesized 2,4-dimethyl pyrrole derived meso-aryl BODIPYs (4.

46-4.53)

display absorptions between 508 nm and 520 nm and in between 533 nm

Table 4 . 6

 46

. Absorption spectral properties of meso-aryl BODIPYs 4.47-4.52 and 4.55-4.60 in DCM at room temperature.

Table 4 .

 4

7. Absorption spectral properties of 2-position (iodine (4.49a and 4.51a), TMS (4.46f), TMS deprotected (4.46g) and π-conjugated (4.66)) substituted derivatives in DCM at room temperature.

3 M -1 cm -1 ]

	4.49a	523	79.5
	4.51a	551	37.56
	4.46f	535	73.01
	4.46g	526	16.76
	4.66	559	82.05

BODIPYs Singlet oxygen quantum yield (ΦΔ) BODIPYs Singlet oxygen quantum yield (ΦΔ)

	BODIPY 4.46	0.01	BODIPY 4.54	0.01
	BODIPY 4.47	0.01	BODIPY 4.55	0.49
	BODIPY 4.48	0.01	BODIPY 4.56	0.01
	BODIPY 4.49	0.01	BODIPY 4.57	0.08
	BODIPY 4.50	0.09	BODIPY 4.58	0.50
	BODIPY 4.51	0.01	BODIPY 4.59	0.02
	BODIPY 4.52	0.32	BODIPY 4.60	0.49

Table 4 . 9 .

 49 Relative singlet oxygen production of for water-soluble derivatives (methylates (4.48a and

	4.56a), carboxylates (4.46b, 4.54b, 4.48b and 4.56b) and etherates (4.46c and 4.54c)), boron-ethynyl
	functionalized (4.46d and 4.50a) and 2-position functionalized (iodine (4.46e, 4.49a, 4.51a) and TMS
	(4.46f,4.51b) derivatives DCM/MeOH (1:1) using RB, ΦΔ = 0.76 as a reference.

Table 4 .

 4 10. Minimum inhibitory concentration (MIC) of BODIPYs tested against S.aureus

	Minimum Inhibitory Concentration (µM)

Table 4 .

 4 11. Minimum inhibitory concentration (MIC) of BODIPYs tested against P. aeruginosa

	Minimum Inhibitory Concentration (µM)

  Rf = 0.32 (DCM/Hex: 90/10); 1 H NMR (400 MHz, CDCl3) δ 9.38 (s, 1H, Ar-H), 8.72 (s, 2H, Ar-H), 6.05 (s, 2H, 2-H, 6-H), 2.57 (s, 6H, 3-CH3, 5-CH3), 1.42 (s, 6H, 1-CH3, 7-CH3). 13 C NMR (101 MHz, CDCl3) δ 159.06, 157.32, 155.92, 142.23, 133.03, 131.44, 129.80, 122.44, 15.59, 14.71. 19 F NMR (377 MHz, CDCl3) δ -

	N yield). M.p. = 177 -182 °C; 145.93 --146.29 (m). 11 B NMR (128 MHz, CDCl3) δ 0.69 (t, J = 32.3 Hz). IR (neat):
	2956, 2922, 2853, 1731, 1546, 1410, 1306, 1183, 1076, 969 cm -1 . UV-Vis (CH2Cl2) λmax
	N	B	N
	F F
	N		N
	N	B	N
	F F

  13 C NMR (101 MHz, CDCl3) δ 156.67, 150.42, 148.52, 142.80, 141.83, 136.43, 131.09, 130.58, 130.09, 128.18, 126.90, 124.98, 121.83, 120.99, 14.83, 14.19.

	1,3,5,7-Tetramethyl-8-(2-quinolinium-yl)-4-bora-3a,4a-diaza-s-indacene (4.49) In accordance with general procedure B as described above using 2,4-dimethylpyrrole
	(2.0 mL, 19.4 mmol) and 9-acridine carboxaldehyde (1.86 g, 9 mmol) and final product
	was obtained and purified by column chromatography (silica gel; DCM/Hex, 9:1, v/v)
	N and removal of solvents followed by recrystallization (DCM/Hex) to yield green solid
	(14% yield). M.p. = 230 -235 °C; Rf = 0.43 (DCM/Hex: 90/10); 1 H NMR (400 MHz,
	B CDCl3) δ 8.28 (d, J = 8.8 Hz, 2H, Ar-H), 7.96 (d, J = 8.7 Hz, 2H, Ar-H), 7.85 -7.78 (m, N N
	F F 2H, Ar-H), 7.55 -7.49 (m, 2H, Ar-H), 5.93 (s, 2H, 2-H, 6-H), 2.63 (s, 6H, 3-CH3, 5-CH3),
	BODIPY 4.49 0.68 (s, 6H, 1-CH3, 7-CH3). 13 C NMR (101 MHz, CDCl3) δ 156.82, 148.85, 142.87,
	139.27, 135.32, 131.70, 130.88, 129.91, 127.46, 125.37, 124.86, 121.85, 14.89, 13.62.
	In accordance with general procedure B as described above using 2,4-dimethylpyrrole 19 F NMR (377 MHz, CDCl3) δ -145.87--146.13. 11 B NMR (128 MHz, CDCl3) δ 0.98 (t, J
	N (2.0 mL, 19.4 mmol) and 2-quinoline carboxaldehyde (1.41 g, 9 mmol) and final product = 32.7 Hz). IR (neat): 3659, 2981, 1965, 1538, 1406, 1299, 1150, 970 cm -1 . UV-Vis
	was obtained and purified by column chromatography (silica gel; DCM/Hex, 8:2, v/v) (CH2Cl2) λmax (ε [x10 3 M -1 cm -1 ]) = 318 (5.20), 365 (17.94), 482 (22.77), 511 (98.28) nm.
	and removal of solvents followed by recrystallization (DCM/Hex) to yield green solid HRMS (APCI): m/z calcd for C27H23BF2N3 [M + H] + : 426.195224, 426.195570 found.
	N (14% yield). M.p. = >300 °C; Rf = 0.51 (DCM/Hex: 90/10); 1 H NMR (400 MHz, CDCl3) N B F F δ 8.33 (d, J = 8.3 Hz, 1H, Ar-H), 8.17 (d, J = 8.4 Hz, 1H, Ar-H), 7.93 (d, J = 8.2 Hz, 1H, 1,3,5,7-Tetramethyl-8-(2-thiazolyl)-4-bora-3a,4a-diaza-s-indacene (4.51)
	Ar-H), 7.81 (ddd, J = 8.4, 7.0, 1.4 Hz, 1H, Ar-H), 7.67 (ddd, J = 8.1, 7.0, 1.1 Hz, 1H, Ar-
	H), 7.53 (d, J = 8.4 Hz, 1H, Ar-H), 5.98 (s, 2H, 2-H, 6-H), 2.57 (s, 6H, 3-CH3, 5-CH3),
	1.22 (s, 6H, 1-CH3, 7-CH3). 13 C NMR (101 MHz, CDCl3) δ 156.63, 154.18, 148.20,
	142.73, 138.92, 137.43, 131.49, 130.62, 129.84, 127.95, 127.80, 127.58, 121.82,
	121.43, 14.83, 14.07. 19 F NMR (377 MHz, CDCl3) δ -145.21--147.31. 11 B NMR (128
	MHz, CDCl3) δ 0.81 (t, J = 32.8 Hz). IR (neat): 3401, 2971, 1596, 1507, 1339, 1185, 971 BODIPY 4.51
	cm -1 . UV-Vis (CH2Cl2) λmax (ε [x10 3 M -1 cm -1 ]) = 318 (12.4), 366 (7.59), 478 (21.22), 508
	(86.52) nm. HRMS (ESI): m/z calcd for C22H20BF2N3 [M + H] + : 376.179507, 376.180084 In accordance with general procedure B as described above using 2,4-dimethylpyrrole
	found. (2.0 mL, 19.4 mmol) and 2-thiazole carboxaldehyde (1.02 g, 9 mmol) and final product
	was obtained and purified by column chromatography (silica gel; DCM/Hex, 8:2, v/v)
	1,3,5,7-Tetramethyl-8-(9-acridinyl)-4-bora-3a,4a-diaza-s-indacene (4.50) and removal of solvents followed by recrystallization (DCM/Hex) to yield green solid
	N (15% yield). M.p. = >300 °C; Rf = 0.37 (DCM/Hex: 90/10); 1 H NMR (400 MHz, CDCl3) δ
	8.00 (d, J = 3.2 Hz, 1H, Ar-H), 7.62 (d, J = 3.2 Hz, 1H, Ar-H), 6.02 (s, 2H, 2-H, 6-H), 2.56 19 F NMR (377 MHz, CDCl3) δ -145.98--146.79. 11 B NMR (128 MHz, CDCl3) δ 0.84 (t, J (s, 6H, 3-CH3, 5-CH3), 1.46 (s, 6H, 1-CH3, 7-CH3). 13 C NMR (101 MHz, CDCl3) δ 160.11, = 32.7 Hz). IR (neat): 3656, 2980, 1588, 1462, 1365, 1187, 969 cm -1 . UV-Vis (CH2Cl2) λmax (ε [x10 3 M -1 cm -1 ]) = 321 (2.93), 348 (5.85), 477 (19.01), 508 (87.61) nm. HRMS N 157.43, 143.41, 143.00, 131.90, 122.25, 121.72, 121.70, 14.77, 13.24. 19 F NMR (377 N B F F MHz, CDCl3) δ -145.22--146.96. 11 B NMR (128 MHz, CDCl3) δ 0.71 (t, J = 32.6 Hz). IR (APCI): m/z calcd for C22H20BF2N3 [M + H] + : 376.179507, 376.179858 found. BODIPY 4.50 (neat): 3116, 2970, 2929, 1692, 1507, 1363, 1186, 966 cm -1 . UV-Vis (CH2Cl2) λmax (ε
	[x10 3 M -1 cm -1 ]) = 322 (3.41), 384 (3.46), 488 (14.62), 520 (59.73) nm. HRMS (APCI):
	m/z calcd for C16H16BF2N3S [M + H]

+ : 332.120180, 332.120779 found.

  C NMR (101 MHz, CDCl3) δ 157.71, 142.96, 142.95, 140.14, 132.80, 129.58, 121.43, 121.25, 33.10, 14.90, 12.14. 19 F NMR (377 MHz, CDCl3) δ -145.17--146.93. 11 B NMR (128 MHz,

	CDCl3) δ 0.73 (t, J = 32.7 Hz). IR (neat): 3151, 2980, 2919, 1690, 1547, 1406, 1151,

  C NMR (101 MHz, CDCl3) δ 166.21, 166.17, 146.96, 145.92, 143.68, 139.48, 132.73, 115.23, 90.80, 90.76, 17.50, 14.62, 9.14, 1.05. IR (neat): 2262, 1576, 1276, 811 cm -1 . HRMS (APCI): m/z calcd for C15H16N3S2 [M + H] + : 302.078016, 302.078557 found. J = 19.3 Hz, 6H, 1-CH3, 7-CH3), 0.20 (s, 9H, 2-TMS). 13 C NMR (101 MHz, CDCl3) δ 158.63, 157.83, 150.75, 144.08, 143.24, 142.88, 138.03, 131.41, 128.94, 123.18, 122.65, 116.00, 101.61, 97.04, 14.77, 13.52, 1.03. 19 F NMR (377 MHz, CDCl3)

	1,3,5,7-Tetramethyl-8-(4-pyridinium-yl-N-(benzyl-4-carboxylate))-4-bora-2,6-Diethyl-1,3,5,7-tetramethyl-8-(4-pyridinium-yl-N-(benzyl-4-carboxylate))-4-1,3,5,7-Tetramethyl -8-(4-quinolinium-yl-N-(benzyl-4-carboxylate))-4-bora-3a,4a-2,6-Diethyl-1,3,5,7-tetramethyl-2,6-diethyl-8-(4-quinolinium-yl-N-(benzyl-4-1,3,5,7-Tetramethyl-8-(N-(4-phenoxybutyl)pyridinium-4-yl)-4-bora-3a,4a-diaza-s-2,6-Diethyl-1,3,5,7-tetramethyl-8-(N-(4-phenoxybutyl)pyridinium-4-yl)-4-bora-1,3,5,7-tetramethyl-8-(4-pyridyl)-4,4-(3-(dimethylamino)prop-1-yn-1-yl)-4-bora-1,3,5,7-tetramethyl-8-(9-acridinyl)-4,4-(3-(dimethylamino)prop-1-yn-1-yl)-4-bora-2-Iodo-1,3,5,7-tetramethyl-8-(4-pyridyl)-4-bora-3a,4a-diaza-s-indacene (4.46e) 2-Iodo-1,3,5,7-tetramethyl-8-phenyl-4-bora-3a,4a-diaza-s-indacene (4.53a) 7.28 (d, J = 4.7 Hz, 2H, Ar-H), 6.05 (s, 1H, 6-H), 2.60 (d, J = 23.0 Hz, 6H, 3-CH3, 5-CH3),
	3a,4a-diaza-s-indacene (4.46b) HOOC bora-3a,4a-diaza-s-indacene (4.54b) HOOC diaza-s-indacene (4.48b) HOOC carboxylate))-4-bora-3a,4a-diaza-s-indacene (4.56b) .Br -.Br -N .Br -N .Br -N HOOC indacene (4.46c) O 3a,4a-diaza-s-indacene (4.54c) O 3a,4a-diaza-s-indacene (4.46d) N 3a,4a-diaza-s-indacene (4.50a) N B N N N I B N N 1.44 (d, δ -146.43--146.17. 11 B NMR (128 MHz, CDCl3) δ 0.57 (t, J = 32.2 Hz). IR (neat): 3044, I N N .Br -.Br -N B N N N N F F F F 2959, 2147, 1733, 1599, 1537, 1309, 1059, 982 cm -1 . UV-Vis (CH2Cl2) λmax (ε [x10 3 M -1 B BODIPY 4.46e BODIPY 4.53a cm -1 ]) = 313 (4.00), 394 (9.06), 505(34.45), 535 (73.01) nm. HRMS (APCI): m/z calcd
	B In accordance with general procedure H as described above using BODIPY 4.46 (0.1g, N N B N N F F B N N F F F F N N In accordance with general procedure H as described above using BODIPY 4.53 (0.1 g, B N N N N for C21H26BF2N3Si [M + H] + : 422.203434, 422.203236 found.
	BODIPY 4.46b F F BODIPY 4.54b BODIPY 4.48b BODIPY 4.56b B N N F F N N BODIPY 4.46d BODIPY 4.50 0.305 mmol) and N-iodosuccinimide (0.085 g, 0.457 mmol) to yield a pinkish-red solid 0.308 mmol) and N-iodosuccinimide (0.086 g, 0.462 mmol) and final product was 1,3,5,7-tetramethyl-8-(2-thiazolyl)-2-trimethylsilyl-4-bora-3a,4a-diaza-s-indacene B F F (81% yield). Analytical data is identical to the literature. [9] M.p. = 200 -205 °C; Rf = 0.67 obtained and purified by column chromatography (silica gel; DCM/Hex, 1:1, v/v) and (4.51b)
	N H In accordance with general procedure E as described above using BODIPY 4.54 (0.1 g, N S S N In accordance with general procedure E as described above using BODIPY 4.48 (0.1 g, In accordance with general procedure E as described above using BODIPY 4.56 (0.1 g, BODIPY 4.46c BODIPY 4.54c In accordance with general procedure, G as described above using BODIPY 4.46 (0.1 (DCM/Hex: 90/10); 1 H NMR (400 MHz, CDCl3) δ 8.79 (s, 2H, Ar-H), 7.28 (d, J = 4.7 Hz, removal of solvents followed by recrystallization (DCM/Hex) to yield pink solid (83% In accordance with general procedure, G as described above using BODIPY 4.50 (0.1 In accordance with general procedure E as described above using BODIPY 4.46 (0.1 g, 0.260 mmol) and 4-(bromomethyl)benzoic acid (0.447 g, 2.08 mmol) the product is 0.265 mmol) and 4-(bromomethyl)benzoic acid (0.455 g, 2.12 mmol) the product is 0.231 mmol) and 4-(bromomethyl)benzoic acid (0.395 g, 1.84 mmol) the product is g, 0.307 mmol, 3-dimethylamino-1-propyne (0.147 mL, 1.38 mmol) and n-BuLi (1.15 mL, 2H, Ar-H), 6.05 (s, 1H, 6-H), 2.60 (d, J = 23.0 Hz, 6H, 3-CH3, 5-CH3), 1.44 (d, J = 19.3 yield). Analytical data is identical to the literature. [10] M.p. = 146 -151 °C; Rf = 0.65 g, 0.234 mmol, 3-dimethylamino-1-propyne (0.112 mL, 1.05 mmol) and n-BuLi (0.88 mL, 0.305 mmol) and 4-(bromomethyl)benzoic acid (0.524 g, 2.44 mmol) the product is obtained further recrystallized from DCM/MeOH and gave red crystals (82% yield). M.p. = 265 -270 °C; 1 H NMR (400 MHz, MeOD) δ 9.33 (d, J = 6.7 Hz, 2H, Ar-H), 8.38 (d, J = 6.7 Hz, 2H, Ar-H), 8.14 (d, J = 8.3 Hz, 2H, Ar-H), 7.63 (d, J = 8.3 Hz, 2H Ar-H), 6.19 (s, 2H, Ar-N + -CH2), 6.10 (s, 2H, 2-H, 6-H), 2.52 (s, 6H, 3-CH3, 5-CH3), 1.47 (s, 6H, 1-CH3, 7-CH3). 13 C NMR (101 MHz, MeOD) δ 167.25, 157.92, 153.53, 146.23, 142.34, 137.91, 133.76, 132.22, 130.54, 129.39, 129.27, 128.51, 122.29, 64.05, 13.95, 13.28. 19 F NMR (377 MHz, MeOD) δ -146.89--147.15. 11 B NMR (128 MHz, MeOD) δ 0.60 (t, J = 31.7 Hz). IR (neat): 3376, 2980, 1708, 1544, 1306, 1155, 1081, 971 cm -1 . UV-Vis (MeOH) λmax (ε [x10 3 M -1 cm -1 ]) = 300 (5.54), 376 (4.83), 481 (12.75), 511 nm (44.18). HRMS (APCI): m/z calcd for C26H25BF2N3O2 + [M + H] + : 460.200705, 460.199798 found. obtained further recrystallized from DCM/MeOH and gave red crystals (75% yield). M.p. = 270 -275 °C; 1 H NMR (400 MHz, MeOD) δ 9.33 (d, J = 6.6 Hz, 2H, Ar-H), 8.37 (d, J = 6.6 Hz, 2H, Ar-H), 8.15 (d, J = 8.3 Hz, 2H, Ar-H), 7.64 (d, J = 8.3 Hz, 2H, Ar-H), 6.12 (s, 2H, Ar-N + -CH2), 2.51 (s, 6H, 3-CH3, 5-CH3), 2.39 (q, J = 7.5 Hz, 4H, 2-CH2, 6-CH2), 1.39 (s, 6H, 1-CH3, 7-CH3), 1.01 (t, J = 7.6 Hz, 6H, 2-CH3, 6-CH3). 13 C NMR (101 MHz, MeOD) δ 168.83, 157.58, 155.81, 147.51, 147.00, 139.31, 138.58, 135.67, 133.78, 131.94, 130.99, 129.87, 65.42, 17.76, 14.87, 12.85, 12.79. 19 F NMR (377 MHz, MeOD) δ -146.59--147.33. 11 B NMR (128 MHz, MeOD) δ 0.62 (t, J = 32.1 Hz). IR (neat): 3313, 2969, 2658, 1677, 1539, 1312, 1173, 1060, 969 cm -1 . UV-Vis (MeOH) λmax (ε [x10 3 M -1 cm -1 ]) = 310 (3.24), 392 (5.40), 504 (12.49), 537 (33.02) nm. HRMS (APCI): m/z calcd for C30H33BF2N3O2 + [M + H] + : 516.263372, 516.264548 found. obtained further recrystallized from DCM/MeOH and gave red crystals (63% yield). M.p. = 245 -250 °C; 1 H NMR (400 MHz, MeOD) δ 9.81 (d, J = 6.0 Hz, 1H, Ar-H), 8.62 (d, J = 9.0 Hz, 1H, Ar-H), 8.46 (d, J = 6.0 Hz, 1H, Ar-H), 8.32 (s, 1H, Ar-H), 8.30 (d, J = 4.3 Hz, 1H, Ar-H), 8.08 (d, J = 8.4 Hz, 2H, Ar-H), 7.48 (d, J = 8.4 Hz, 2H, Ar-H), 6.59 (s, 2H, Ar-N + -CH2), 6.20 (s, 2H, 2-H, 6-H), 2.59 (s, 6H, 3-CH3, 5-CH3), 1.21 (s, 6H, 1-CH3, 7-CH3). 13 C NMR (101 MHz, MeOD) δ 168.78, 159.70, 155.16, 151.98, 143.67, 143.19, 139.69, 139.04, 138.47, 133.40, 131.85, 131.44, 130.98, 128.83, 128.30, 124.85, 123.73, 121.23, 62.32, 14.75, 14.59. 19 F NMR (377 MHz, MeOD) δ -146.76--147.03. 11 B NMR (128 MHz, MeOD) δ 0.75 (t, J = 31.7 Hz). IR (neat): 2973, 2159, 1705, 1542, 1307, 1191, 1072, 973 cm -1 . UV-Vis (MeOH) λmax (ε [x10 3 M -1 cm -1 ]) = 322 (8.94), 394 (3.76), 481 (15.98), 510 (37.31) nm. HRMS (APCI): m/z calcd for C30H27BF2N3O2 + [M + H] + : 510.216420, 510.214860 found. obtained further recrystallized from DCM/MeOH and gave red crystals (68% yield). M.p. 7.49 (d, J = 8.4 Hz, 2H, Ar-H), 6.60 (s, 2H, Ar-N + -CH2), 2.58 (s, 6H, 3-CH3, 5-CH3), 2.37 (q, J = 7.5 Hz, 4H, 2-CH2, 6-CH2), 1.13 (s, 6H, 1-CH3, 7-CH3), 0.99 (t, J = 7.5 Hz, 6H, 2-CH3, 6-CH3). 13 C NMR (101 MHz, MeOD) δ 159.70, 155.09, 151.85, 143.68, 139.72, 138.42, 133.37, 131.77, 131.72, 131.45, 130.97, 130.15, 128.80, 128.10, 124.81, 123.77, 121.26, 62.40, 47.95, 32.76, 23.74, 14.58, 14.43. 19 F NMR (377 MHz, MeOD) δ -146.21--146.53. 11 B NMR (128 MHz, MeOD) δ 0.75 (t, J = 31.9 Hz). IR (neat): 3313, 2969, 1944, 1677, 1539, 1312, 1173, 1060, 969 cm -1 . UV-Vis (MeOH) λmax (ε [x10 3 M -1 cm -1 ]) = 322 (9.66), 408 (5.73), 506 (18.55), 536 (30.80) nm. HRMS (APCI): m/z calcd for C34H35BF2N3O2 + [M + H] + : 566.279085, 566.277708 found. In accordance with general procedure F as described above using BODIPY 4.46 (0.1 g, 0.305mmol) and 4-bromobutyl phenyl ether (0.559 g, 2.44 mmol) the product is obtained further recrystallized from DCM/MeOH and gave red crystals (70% yield). M.p. = 240 -245 °C; 1 H NMR (400 MHz, CDCl3) δ 9.78 (d, J = 6.7 Hz, 2H, Ar-H), 8.00 (t, J = 9.7 Hz, 2H, Ar-H), 7.35 -7.26 (m, 2H, Ar-H), 6.96 (t, J = 7.4 Hz, 1H, Ar-H), 6.87 (d, J = 7.8 Hz, 2H, Ar-N + -CH2), 6.06 (s, 2H, 2-H, 6-H), 5.34 (t, J = 7.5 Hz, 2H, Ar-H), 4.07 (t, J = 5.6 Hz, 2H, Ar-H), 2.57 (s, 6H, 3-CH3, 5-CH3), 2.37 (dd, J = 14.7, 7.5 Hz, 2H, Ar-H), 1.94 (dd, J = 13.7, 5.8 Hz, 2H, Ar-H), 1.41 (d, J = 4.5 Hz, 6H, 1-CH3, 7-CH3). 13 C NMR (101 MHz, CDCl3) δ 158.77, 158.38, 146.03, 141.64, 132.24, 129.68, 129.27, 128.61, 123.31, 123.02, 121.23, 114.42, 66.85, 62.02, 29.61, 25.48, 15.51, 14.82. 19 F NMR (377 MHz, CDCl3) δ -145.80--146.05. 11 B NMR (128 MHz, MeOD) δ 0.75 (t, J = 31.9 Hz). IR (neat): 2955, 1985, 1640, 1543, 1296, 1150, 1080, 970 cm -1 . UV-Vis (CH2Cl2) λmax (ε [x10 3 M -1 cm -1 ]) = 307 (5.76), 367 (6.5), 476 (17.27), 506 (61.94) nm. HRMS (APCI): m/z calcd for C28H31BF2N3O + [M + H] + : 474.252774, 474.251830 found. 6.94 (t, J = 6.9 Hz, 1H, Ar-H), 6.87 (d, J = 7.8 Hz, 2H, Ar-N + -CH2), 5.38 (t, J = 7.3 Hz, 2H, Ar-H), 4.06 (t, J = 5.7 Hz, 2H, Ar-H), 2.54 (s, 6H, 3-CH3, 5-CH3), 2.37 (dd, J = 14.8, 7.3 Hz, 2H, Ar-H), 2.28 (q, J = 7.5 Hz, 4H, 2-CH2, 6-CH2), 1.92 (d, J = 12.1, 5.9 Hz, 2H, Ar-H), 1.67 (s, 2H, Ar-H), 1.30 (s, 6H, 1-CH3, 7-CH3), 0.97 (t, J = 7.5 Hz, 6H, 2-CH3, 6-CH3). 13 C NMR (101 MHz, CDCl3) δ 158.54, 157.15, 154.68, 146.11, 146.05, 136.77, 134.90, 130.97, 129.77, 128.95, 121.28, 114.55, 66.97, 61.97, 29.75, 25.64, 17.19, 1.228 mmol) and final product was obtained and purified by column chromatography 5 Hz, 2H, Ar-H), 7.51 (dd, J = 4.4, 1.6 Hz, 2H, Ar-H), 6.14 (s, 2H, 2-H, 6-H), 3.22 (s, 4H, CH2), 2.77 (s, 6H, 3-CH3, 5-CH3), 2.30 (s, 12H, N-CH3), 1.44 (s, 6H, 1-CH3, 7-CH3). 13 C NMR (101 MHz, MeOD) δ 156.31, 150.21, 145.24, 140.93, 138.24, 128.59, 124.66, 122.06, 43.21, 15.47, 13.92. 11 B NMR (128 MHz, MeOD) δ -13.34 (s). IR (neat): 2926, 2163, 1653, 1598, 1305, 1176, 1093, 978 cm -1 . UV-Vis (MeOH) λmax (ε [x10 3 M -1 cm -1 ]) = 308 (6.69), 356 (5.22), 474 (13.41), 504 (57.21) nm. HRMS (APCI): m/z calcd for C28H34BN5 0.938 mmol) and final product was obtained and purified by column chromatography H), 7.91 (d, J = 8.6 Hz, 2H, Ar-H), 7.84 -7.80 (m, 2H, Ar-H), 7.54 -7.49 (m, 2H, Ar-H), 5.99 (s, 2H, 2-H, 6-H), 3.59 (s, 4H, CH2), 2.80 (s, 6H, 3-CH3, 5-CH3), 2.61 (s, 12H, N-CH3), 0.67 (s, 6H, 1-CH3, 7-CH3). 13 C NMR (101 MHz, CDCl3) δ 156.01, 148.74, 141.13, (DCM/Hex: 90/10); 1 H NMR (400 MHz, CDCl3) δ 7.51 -7.47 (m, 3H, Ar-H), 7.27 -7.24 (m, 2H, Ar-H), 6.04 (s, 1H, 6-H), 2.63 (d, J = 23.0 Hz, 6H, 3-CH3, 5-CH3), 1.37 (s, 6H, 1-Hz, 6H, 1-CH3, 7-CH3). 2-Iodo-1,3,5,7-tetramethyl-8-(2-quinolinium-yl)-4-bora-3a,4a-diaza-s-indacene BODIPY 4.51b In accordance with general procedure I as described above using BODIPY 4.51a (25 CH3, 7-CH3). 1,3,5,7-tetramethyl-8-(4-pyridyl)-2-trimethylsilyl-4-bora-3a,4a-diaza-s-indacene mg, 0.055 mmol), CuI (0.001 g, 0.005 mmol), Pd(PPh3)Cl2 (0.0014 g, 0.002 mmol) and (4.49a) TMSA (530 µl, 0.54mmol) and final product was obtained and purified by column (4.46f) 139.31, 135.48, 130.73, 129.85, 129.63, 127.35, 125.17, 124.77, 122.26, 48.51, 43.09, chromatography (silica gel; DCM/Hex, 2:3, v/v) and removal of solvents followed by N 29.70, 16.33, 13.70. 11 B NMR (128 MHz, CDCl3) δ -0.96 (s). IR (neat): 2923, 2163, 1653, recrystallization (DCM/Hex) to yield pink solid (65% yield). M.p. = 155 -160 °C; Rf = N 1548, 1361, 1176, 1094, 977 cm -1 . UV-Vis (CH2Cl2) ) λmax (ε [x10 3 M -1 cm -1 ]) = 310 (5.69), 0.65 (DCM/Hex: 90/10); 1 H NMR (400 MHz, CDCl3) δ 8.02 (d, J = 3.3 Hz, 1H, Ar-H), 14.62, 13.05, 12.95. 19 F NMR (377 MHz, CDCl3) δ -145.42--145.68. 11 B NMR (128 MHz, CDCl3) δ 0.59 (t, J = 32.4 Hz). IR (neat): 2964, 1976, 1635, 1540, 1312, 1157, [M + H] + : 452.298501, 452.298621 found. 362 (12.41), 477 (12.71), 507 (54.73) nm. HRMS (APCI): m/z calcd for C36H38BN5 [M + H] + : calcd. 552.329929, found 552.330228. B N N F F I B N 7.64 (d, J = 3.3 Hz, 1H, Ar-H), 6.06 (s, 1H, 6-H), 2.60 (d, J = 21.6 Hz, 6H, 3-CH3, 5-CH3), N F F TMS 1.49 (d, J = 14.2 Hz, 6H, 1-CH3, 7-CH3), 0.21 (s, 9H, 2-TMS). 13 C NMR (101 MHz, 1075, 970 cm -1 . UV-Vis (CH2Cl2) λmax (ε [x10 3 M -1 cm -1 ]) = 307 (5.76), 367 (6.5), 476 BODIPY 4.49a BODIPY 4.46f CDCl3) δ 158.41, 155.31, 153.71, 148.23, 144.72, 143.1, 138.54, 137.38, 132.01, (17.27), 506 (61.94) nm. HRMS (APCI): m/z calcd for C32H39BF2N3O + [M + H] + : 130.51, 129.51, 128.10, 127.48, 122.37, 121.48, 84.27, 16.22, 14.6,1.02. 19 F NMR (377 530.315440, 530.314255 found In accordance with general procedure H as described above using BODIPY 4.49 (0.1 g, In accordance with general procedure I as described above using BODIPY 4.46e (25 MHz, CDCl3) δ -144.99--146.50. 11 B NMR (128 MHz, CDCl3) δ 0.60 (t, J = 31.9 Hz). IR
	0.265 mmol) and N-iodosuccinimide (74.5 g, 0.397 mmol) and final product was mg, 0.055 mmol), CuI (0.001 g, 0.005 mmol), Pd(PPh3)Cl2 (0.0014 g, 0.002 mmol) and (neat): 2924, 2147, 15421, 1537, 1309, 1061, 980 cm -1 . UV/Vis (CDCl3 λmax (ε [x10 3 M -
	obtained and purified by column chromatography (silica gel; DCM/Hex, 1:1, v/v) and TMSA (530 µl, 0.54mmol) and final product was obtained and purified by column 1 cm -1 ]) = 309 (2.55), 406 (5.34), 520 (19.36), 551 (37.56) nm. HRMS (APCI): m/z calcd
	for C19H24BF2N3SSi [M + H]

= 184 -189 °C; 1 H NMR (400 MHz, MeOD) δ 9.80 (d, J = 6.0 Hz, 1H, Ar-H), 8.62 (d, J = 8.8 Hz, 1H, Ar-H), 8.43 (d, J = 5.8 Hz, 1H, Ar-H), 8.33 (d, J = 8.3 Hz, 1H, Ar-H), 8.30 (d, J = 8.9 Hz, 1H, Ar-H), 8.10 (d, J = 8.3 Hz, 2H, Ar-H), 8.05 (t, J = 7.7 Hz, 1H, Ar-H), In accordance with general procedure F as described above using BODIPY 4.54 (0.1 g, 0.260 mmol) and 4-bromobutyl phenyl ether (0.476 g, 2.08 mmol) the product is obtained further recrystallized from DCM/MeOH (72% yield). M.p. = 210 -215 °C; 1 H NMR (400 MHz, CDCl3) δ 9.85 (d, J = 6.3 Hz, 2H, Ar-H), 8.00 (d, J = 6.4 Hz, 2H, Ar-H), (silica gel; DCM/MeOH, 9.9:0.1, v/v) and removal of solvents followed by recrystallization (DCM/Hex/MeOH) to yield pink solid (45% yield). M.p. = 155 -160 °C; Rf = 0.35 (DCM/MeOH: 99/1); 1 H NMR (400 MHz, MeOD) δ 8.75 (dd, J = 4.5, 1.(silica gel; DCM/MeOH, 9.8:0.2, v/v) and removal of solvents followed by recrystallization (DCM/Hex/MeOH) to yield pink solid (30% yield). M.p. = 187 -192 °C; Rf = 0.32 (DCM/MeOH: 99/1); 1 H NMR (400 MHz, CDCl3) δ 8.28 (d, J = 8.8 Hz, 2H, Ar-19 F NMR (377 MHz, CDCl3) δ -146.17--146.43. 11 B NMR (128 MHz, CDCl3) δ 0.57 (t, J = 32.2 Hz). IR (neat): 2923, 2024, 1716, 1598, 1532, 1157, 977 cm -1 . HRMS (APCI): m/z calcd for C18H17BF2IN3 [M + H] + : 452.060437, 452.060466 found. removal of solvents followed by recrystallization (DCM/Hex) to yield pink solid (42% yield). M.p. = 220 -225 °C; Rf = 0.47 (DCM/Hex: 90/10); 1 H NMR (400 MHz, CDCl3) δ 8.35 (d, J = 8.3 Hz, 1H, Ar-H), 8.16 (d, J = 8.6 Hz, 1H, Ar-H), 7.94 (d, J = 7.8 Hz, 1H, 19 F NMR (377 MHz, CDCl3). 11 B NMR (128 MHz, CDCl3) δ 0.71 (t, J = 32.4 Hz). IR (neat): 2921, 1970, 1735, 1618, 1552, 1185, 970 cm -1 . HRMS (APCI): m/z calcd for C19H18BF2IN2 [M + H] + : 449.050651, 449.050778found. chromatography (silica gel; DCM/Hex, 3:7, v/v) and removal of solvents followed by recrystallization (DCM/Hex) to yield pink solid (78% yield). M.p. = 175 -180 °C; Rf = 0.63 (DCM/Hex: 90/10); 1 H NMR (400 MHz, CDCl3) δ 8.79 (d, J = 2.5 Hz, 2H, Ar-H), + : 428.159827, 428.159687 found.

,5,7-tetramethyl-8-(4-pyridyl)-4-bora-3a,4a-diaza-s-indacene)buta-1,3-diyn- 1-yl)-1,3,5,7-tetramethyl-8-(4-pyridyl)-4-bora-3a,4a-diaza-s-indacene (4.66)

	B F F N N N 2-((1,3BODIPY 4.66 B N N F F N	B F F N N N
	BODIPY derivative 4.46g (1 eq.), CuI (0.15 eq.) and Pd(PPh3)Cl2 (0.2 eq.) was dried
	under vacuum for about 4-5 hours and then were dissolved in dry THF:TEA(2:1) under
	inert Ar atmosphere. After 30 min BODIPY 4.46e (1.5 eq.) was dissolved in 1 ml dry
	THF and was added to the previous mixture and the mixture was refluxed for 20 h at 66
	ºC. The mixture was then filtered and washed with a solution of saturated NaHCO3 (2 x
	50 ml) followed by washing with deionized water (2 x 50 ml). The resulting solution is
	dried over anhydrous sodium sulphate and evaporated in a rotavapor. The residue was
	purified by flash column chromatography eluting with DCM:ethyl acetate (9:1 ) to yield
	the pure product as a purple crystalline solid (42% yield). M.p. = >300 °C; Rf = 0.21
	(DCM/Ethyl acetate: 90/10); 1 H NMR (400 MHz, CDCl3) δ 8.85 (s, 4H, Ar-H), 7.42 (s,
	13 C NMR (101 MHz, CDCl3) δ 158.86, 4H, Ar-H), 6.09 (s, 2H, 2-H, 6'-H), 2.65 (s, 6H, 3-CH3, 5-CH3), 2.59 (s, 6H, 1-CH3, 7-
	155.49, 153.76, 148.09, 144.61, 142.80, 138.54, 137.48, 131.89, 130.67, 129.72, CH3), 1.47 (s, 6H, 3'-CH3, 5'-CH3), 1.42 (s, 6H, 1'-CH3, 7'-CH3). 13 C NMR (101 MHz,
	127.86, 127.50, 122.38, 121.53, 35.21, 16.22, 14.6. 19 F NMR (377 MHz, CDCl3) δ -CDCl3) δ 159.00, 159.00, 157.25, 157.25, 156.01, 156.01, 142.39, 133.01, 131.46,
	146.29--146.03. 11 B NMR (128 MHz, CDCl3) δ 0.56 (t, J = 32.2 Hz). IR (neat): 3044, 131.44, 129.91, 129.91, 122.28, 122.28, 15.60, 14.71. 19 F NMR (377 MHz, CDCl3) δ -
	2959, 2147, 1733, 1599, 1537, 1309, 1059, 982 cm -1 . UV-Vis (CH2Cl2) λmax (ε [x10 3 M -1 145.97--146.22. 11 B NMR (128 MHz, CDCl3) δ 0.52 (t, J = 31.9 Hz). IR (neat): 2961,
	cm -1 ]) = 311 (0.94), 383 (1.79), 494 (5.81), 526 (16.76) nm. HRMS (APCI): m/z calcd for 1591, 1410, 1321, 1159, 1031, 980 cm -1 . UV-Vis (CH2Cl2) λmax (ε [x10 3 M -1 cm -1 ]) = 328
	C19H20BF2N3 [M + H] + : 350.163823, 350.163410 found. (9.27), 412 (12.59), 520 (31.47), 559 (82.05) nm. HRMS (APCI): m/z calcd for
	C40H34B2F4N6 [M + H] + : 677.299074, 677.298843 found.

  The microplate was incubated at 37°C for 5h with white light (total fluence: 25J/cm 2 ) or in the dark. After the microplate was left at 37°C for 24h.The inhibition of biofilm formation was determined by Crystal violet Assay (biofilm reduction compared to the untreated control). The microplate was washed with distilled water. Then 125μL of 0.1% crystal violet was added in the microplate well for 15 min at RT. After incubation, the microplate was washed with distilled water and dried for 2 h. The optical density will be read at 595 nm. Obtained optical density (OD) for untreated control corresponded to 100% of biofilm. Obtained OD in other conditions (32MIC to 1MIC) were compared to untreated control to show the concentration of BODIPY 4.57: Disorder of the quinolinyl fragment was the correct model -an equivalent solution in Cc was unstable and gave 50:50 orientation of the two fragments at any rate. A loose ISOR restraint was used to constrain prolate behavior in C9, the terminal C1-Omitted ref. 0 1 5 and 6 1 6 due to stark disagreement with the model. BODIPY 4.48a: No special refinement necessary.BODIPY 4.46b: A squeeze model was chosen -the refinement was highly unstable when concerned with splitting individual solvent molecules, however the main fragment was well-ordered. Intermolecular hydrogen bonding which sits between O67 and its centrosymmetry partner indicate that this compound is at least 1/8 th protonated and that a disorder model is appropriate, given the inability to appropriately model counteranions. As such, the exact protonation state of this compound is unknown.

	The inhibition of biofilm formation was determined on bacterial cultures (Staphylococcus aureus CIP 76.25) by micro method in 96-well plates. Briefly, bacterial cultures in log-phase were diluted in TS broth and glucose (25mM) at a concentration equivalent to 4x10^6 CFU/mL. Serial dilutions of BODIPYs were performed in TS broth and glucose (25mM) to a final concentration from MIC 35µM to MIC 0.5µM and 100 μL of each dilution were added in the microplate wells. Then, 100 μl of diluted bacterial culture were added in each well. Two different plates were prepared: one plate for the dark condition and a Then, 150μL of 33% acetic acid were added for 20 min at RT in the microplate wells to solubilize the biofilm. The optical density will be read at 595nm. Three independent experiments were performed. General procedure for evaluation of biofilm eradication: Biofilm eradication was evaluated on bacterial cultures in 24-well plates. Briefly, bacterial cultures in log-phase were diluted in TS broth including glucose (25mM) for obtaining a concentration equivalent to 2x10^6 CFU/mL. 1mL of this diluted bacterial culture was added in each Three controls were included: a bacterial growth control with no molecule added, a negative control with culture broth without bacterial inoculum and no molecule and a positive control with bleach 3% added to the bacterial inoculum. The microplate was incubated at 37°C for 5h with white LED light (total fluence: 25J/cm²) or in the dark. Then, 500 µL of 2-fold concentrated TS broth and glucose (25mM) were added in each well and the microplate was incubated at 37°C for 24h. Biofilm eradication was determined by Crystal violet Assay (biofilm reduction compared to the untreated control). The microplate was washed with distilled water. Then, 125μL of 0.1% crystal violet were added in the microplate well for 15 min at RT. After incubation, the microplate was washed with distilled water and dried for 2 h. Then, 150µL of 33% acetic acid were added for 20 min at RT in the microplate wells to solubilize the BODIPY 4.59: No special refinement necessary. crystal violet. bound methyl group. BODIPY 4.60:

second plate for light irradiation. Three controls were included; a bacterial growth control with no molecule added, a negative control with TS broth and glucose without bacterial inoculum and no molecule and a positive control with javel 3% added to the bacterial inoculum.

well. The microplate was incubated at 37°C, 100rpm for 24h in dark condition and in aerobic conditions. After this time, culture broth was removed. In parallel, serial dilutions of different molecules were performed in PBS to a final concentration from 32MIC to 1MIC and 500µL of each dilution was added in the microplate wells. Two different plates were prepared: one plate for the dark condition and a second plate for light irradiation. BODIPY 4.48b: No special refinement necessary.

Table 5 . 1 .

 51 Details of crystal structure determinations.

  4-bora-3a,4a-diaza-

	BODIPY		4.48b		4.54b		4.56b		4.52a
	Chemical Name	1,3,5,7-Tetramethyl-2,6-diethyl-8-	1,3,5,7-Tetramethyl-2,6-diethyl-	1,3,5,7-Tetramethyl-8-(4-pyridyl-	(Z,Z)-2,5-Bis(2-
			(4-pyridyl-2-(benzyl-4-carboxylic	8-(4-quinolinium)-yl-2-(benzyl-4-	yl-2-(benzyl-4-carboxylate))-4,4-	thiazolylmethanylidene)-
			acid))-4,4-difluoro-4-bora-3a,4a-	carboxylic acid))-4,4-difluoro-4-	difluoro-4-bora-3a,4a-diaza-s-	3-ethyl-4-methylpyrrole
			diaza-s-indacene	bora-3a,4a-diaza-s-indacene	indacene
										s-indacene
	Chemical
	Formula		C30H33BBrF2N3O2	C38H43BBrF2N3O4	C26H32.12BCl0.12F2N3O6		C15H15N3S2
	Crystal System	monoclinic		orthorhombic	triclinic		monoclinic
	Space Group	P 21/c		P 212121	P -1		P 21/c
	Chemical Formula Description Crystal needle	C23H26BF2N3Si	plate	C20H18BF2N3	C40H34B2F4N6 needle	C23H24BBrF2N3O0.5 plate	C32H41BBrF2N3O2
								0
	Crystal System Crystal color	red	triclinic	monoclinic orange	orthorhombic red	monoclinic red	monoclinic
	Space Group a (Å) b (Å)	10.0239(3) 29.6079(9)	P1	P21/c 8.7972(3) 12.7756(4)	Pna21 11.0357(5) 20.6326(9)	C 2/c	13.3172(11) 10.2096(8)	P 21/c
	c (Å)		9.8588(3)		32.3590(11)	24.0869(10)		22.0316(17)
	Crystal Description ⍺ (°) 90	needle	90	needle	oblong 104.197(2)	oblong	90	oblong
	β (°)		104.3361(14)		90		91.229(2)		102.2850(18)
	Crystal color γ (°)	90	red	90	red	Purple-violet 100.238(3)	red	90	red
	a (Å) Volume (Å 3 )		2834.85(15)	8.7870(3)	14.6509(18) 3636.8(2)	20.7649(5) 5220.1(4)	23.4395(8) 2926.9(4)	19.4295(15)
	b (Å) Crystal	Size	10.9761(4) 0.430 ⨉ 0.104 ⨉ 0.065	11.5401(13) 0.388 ⨉ 0.181 ⨉ 0.010	7.0387(2) 0.420 ⨉ 0.097 ⨉ 0.034	7.3588(3)	18.5995(15) 0.640 ⨉ 0.387 ⨉ 0.156
	c (Å) (mm 3 )			11.6926(4)		10.4773(12)	23.8366(6)	24.9912(8)	8.4588(7)
	⍺ (°) Density (a.u.)	1.397	11	1.341	90	90 1.364	90	1.368	90
	β (°) μ (mm -1 )		2.354	10	1.982	105.490(3)	90 0.999	91.5314(17) 0.357	93.377(4)
	γ (°) Absorption		multi-scan	11	90 multi-scan	90 multi-scan	90	multi-scan	90
	Volume (Å 3 ) Correction		1118.21(7)		1707.1(3)	3483.91(16)	4309.1(3)		3051.5(4)
	Crystal Size (mm 3 ) Tmin, Tmax 0.5741, 0.7531	0.43 x 0.11 x 0.08 0.5723, 0.7531 0.27 x 0.15 x 0.11	0.129 x 0.071 x 0.5791, 0.7531	0.256 ⨉ 0.081 ⨉ 0.6788, 0.7454 0.326 ⨉ 0.220 ⨉ 0.042
	Temp (K)		100(2)	mm3	100(2)	0.033 100(2)	0.020	100(2)
	Density (a.u.)		1.251		1.359	1.328	1.477		1.368
	λ (Å) μ (mm -1 ) Radiation type	1.54178 CuK\a	1.182	1.54178 0.096 CuK\a	1.54178 0.773 CuK\a	2.895	0.71073 MoK\a	1.393
	Absorption Correction	Semi-empirical from	Semi-empirical from	Semi-empirical from	multi-scan	multi-scan
	Measured		53661	equivalents	equivalents 26430	equivalents 86688		39541
	Reflections
	Tmin, Tmax		0.5545, 0.7533		0.5917, 0.7533	0.6153, 0.7533	0.5064, 0.7531	0.5525, 0.7461
	θmax		68.416		68.361	68.438		26.559
	Temp (K) Scan Mode		ω & φ	100.01	100(2) ω & φ	100(2) ω & φ	100(2)	ω & φ	100(2)
	λ (Å) R1 (all)		0.0331	1.54178	0.71073 0.0518	1.54178 0.0837	1.54178	0.0919	0.71073
	Radiation type R1 (I > 2σ)	0.0318	CuK\a	MoK\a 0.0434	CuK\a 0.0622	CuK\a	0.0701	MoK\a
	Measured Reflections wR2 (all) 0.0913	18442	22677 0.1115	53878 0.1966	18075	0.1699	75140
	θmax wR2 (I > 2σ)		0.0904	69.923	26.191 0.1064	69.981 0.1776	68.399	0.1559	30.499
	Scan Mode Goodness of Fit	1.057	ω & φ	1.016	ω & φ	ω & φ 1.066	ω & φ	1.128	ω & φ
	R1 (all) H-atoms ref.	mixed	0.0465	0.0912 mixed	0.0423 constr	0.0681	constr	0.1222
	R1 (I > 2σ)			0.0457		0.0553	0.0359	0.0625		0.0875
	Independent		5183		6585		19030		6082
	wR2 (all) Reflections			0.1299		0.1254	0.0935	0.1980		0.2990
	wR2 (I > 2σ)		0.1290		0.1095	0.0893	0.1926		0.2669
	Goodness of Fit Independent	4903	1.035	5739	1.069	1.025 13396	1.042	4733	1.192
	Reflections (I >
	H-atoms ref. 2σ)		mixed		mixed	mixed	mixed		mixed
	Independent Reflections Max and Min 0.417, -0.516	4177	3400 0.306, -0.275	6502 1.196, -0.364	3939	0.521, -0.696	9270
	residual
	Independent Reflections (I Data / 5183 / 376 / 0	4350	8730 6585 / 398 / 0	6560 19030 / 1241 / 0	3467	6801 6082 53 / 154
	> 2σ) Parameters	/
	Restraints
	Max and Min residual	0.397 and -0.370		0.286 and -0.296	0.262 and -0.167	1.561, -0.832	2.946, -1.580
	Data / Parameters /	4177 / 278/0		3400 /240/0	6502 /478/1	3939 / 285 / 3	9270 / 383 / 3
	Restraints

. I would not have made it this far without you. Hope I made you all proud.

This project has received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Sklodowska-Curie grant agreement n°764837.

Chapter 5.

Experimentation

After reaction overnight, the pH increased to the range of 6.8-7.2. Then, the resulting conjugate was dialyzed against deionized water for three days. The purified conjugate solution was lyophilized for two days.

General Procedure B: BODIPYs (4.46-4.53): 2,4-Dimethylpyrrole (2 eq.) and the appropriate benzaldehyde (1 eq.) were dissolved in anhydrous DCM (150 mL) under argon. Trifluoracetic acid (TFA) (0.2 mL) was added, and the reaction mixture was stirred at room temperature under constant stirring for 12 h. When the aldehyde was consumed (monitored by TLC), 3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) (9 mmol) was added to the previous solution at room temperature. After stirring for 4 h at room temperature, triethylamine (TEA) (1 eq.) and boron trifluoride etherate (BF3•OEt2) (1 eq.)

was added dropwise at 0 °C over a period of 10 min, and the mixture was stirred at room temperature for 3 h. The mixture was then filtered and washed with a solution of saturated NaHCO3 followed by washing with deionized water. The resulting solution is dried over anhydrous NaSO4 and evaporated in a rotavapor. The residue was purified by flash column chromatography eluting with DCM:Hexane solvent systems (varying for different derivatives) to yield the pure product as an orange crystalline solid.

General Procedure C: BODIPYs (4.54-4.60): 3-Ethyl-2,4-dimethylpyrrole (2 eq.) and the appropriate benzaldehyde (1 eq.) were dissolved in anhydrous DCM (150 mL) under argon. Trifluoracetic acid (TFA) (0.2 mL) was added, and the reaction mixture was stirred at room temperature under constant stirring for 12 h. When the aldehyde was consumed (monitored by TLC), DDQ (9 mmol) was added to the previous solution at room temperature. After stirring for 4 h at room temperature, TEA (1 eq.) and BF3•OEt2 (1 eq.) was added dropwise at 0 °C over a period of 10 min, and the mixture was stirred at room temperature for 3 h. The mixture was then filtered and washed with a solution of saturated NaHCO3 followed by washing with deionized water. The resulting solution is dried over anhydrous sodium sulphate and evaporated in a rotavapor. The residue was purified by flash column chromatography eluting with DCM:Hexane solvent system (varying for different derivatives) to yield the pure product as a red crystalline solid.

General Procedure D: BODIPYs (4.46a, 4.54a, 4.48a and 4.56a): Previously synthesized BODIPY (1 eq.) were dissolved in 4mL anhydrous dry acetonitrile (4 mL), and methyl iodide (1 eq.) was added to the above mixture which was then refluxed for mixture was then filtered and washed with a solution of saturated NaHCO3 followed by washing with deionized water. The resulting solution is dried over anhydrous sodium sulphate and evaporated in a rotavapor. The residue was purified by flash column chromatography eluting with Hexane:DCM (1:2) to yield the pure product as a pinkishred crystalline solid.

General Procedure I: BODIPYs (4.46f and 4.51b) Sonogashira cross-coupling:

Previously synthesized BODIPY (1 eq.), CuI (0.1 eq.) and Pd(PPh3)Cl2 (0.05 eq.) were dissolved in 5 mL dry THF:TEA(4:1) at room temperature under inert atmosphere of argon. After stirring for 30 min trimethylsilyl acetylene (TMSA) (10 eq.) was added under argon. The reaction mixture was refluxed for 20 h. The mixture was then filtered and washed with a solution of saturated NaHCO3 followed by washing with deionized water.

The resulting solution is dried over anhydrous sodium sulphate and evaporated in a rotavapor. The residue was purified by flash column chromatography eluting with Hexane:Ethyl acetate (9:1) to yield the pure product as a pinkish-orange crystalline solid.

5-(4-Acetamidophenyl)-10,15,20-tris(4-pyridyl)porphyrin (2.22)

2.22

Porphyrin 2.22 was synthesized using a pyrrole (0.832 mL, 12.0 mmol), 4pyridylcarboxaldehyde (0.847 mL, 9.0 mmol), 4-acetamidobenzaldehyde (489.3 mg, 3.0 mmol) and propionic acid (20 mL). Purification by column chromatography (silica gel; CHCl3/EtOH, 1:0.1, v/v) and removal of solvents followed by recrystallization (CHCl3/MeOH) gave the title compound as purple crystals (4% yield). Analytical data is identical to the literature. [3] M.p. = >300 °C; Rf = 0.54 (CHCl3/EtOH: 90/10); UV-Vis In accordance with general procedure C as described above using 3-ethyl- In accordance with general procedure D as described above using BODIPY 4.46 (0.1 g, 0.305 mmol) and methyl iodide (0.018 mL, 0.305 mmol) the product is obtained further recrystallized from DCM/MeOH gave red crystals (81% yield). Analytical data is identical to the literature. [7] M.p. = 250 -255 °C; In accordance with general procedure D as described above using BODIPY 4.54 (0.1 g, 0.260 mmol) and methyl iodide (0.016 mL, 0.260 mmol) the product is obtained further recrystallized from DCM/MeOH and gave red crystals (73% yield). Analytical data is identical to the literature. [8]

Experimental for X-ray crystallographic structure elucidations

Compounds were crystallized from slow evaporation of methanol (5) or dichloromethane (14). Individual single crystals were mounted on a nylon loop using crystallographic oil, and placed in a stream of N2 at the temperature indicated in 8. [11,12] Reflections were integrated and multi-scan absorption corrections performed using Bruker APEX3 software suite, and equivalent reflections merged with XPREP. Structure solution was performed with SHELXT (2018/2) and refinement performed with SHELXL (2018/3) in the Shelxle GUI.

All non-H atoms were refined anisotropically, with C-bound H-atoms constrained to idealized positions, and N-and O-bound H-atoms to geometric constraints, where appropriate. H atoms were refined with riding isotropic thermal parameters. In each case, aryl rotation was determined with MPLA instructions in ShelXL to obtain precise error margins.

Images were prepared using CrystalMaker software (CrystalMaker Ltd.).

5.4.1.

Additional refinement details for each compound

SUMMARY

Photodynamic therapy (PDT) has been used as a minimally invasive therapeutic tool in clinical settings against malignant cells and infected tissue. The cytotoxicity is induced by a light source potentially exerting a selective activity against the infections. This approach has found potential anti-microbial uses in recent times against all types of microorganisms, such as Gram-positive and Gram-negative bacteria, fungi, parasites and even viruses. Antimicrobial Photodynamic therapy (aPDT) provides a possible way to eliminate diseases causing microbes efficiently, cost-effectively, and more importantly, without the associated mechanism of resistance. This manuscript describes inclusive study into synthetic and post-synthetic advances made towards enhancing the efficiency of photosensitizers (PSs) for this therapy. More explicitly, it provides an insight to making a PS aPDT compatible by combining it with a drug delivery system or synthetically altering the molecular skeleton of the PS with functionalities which induces better potency towards the site of infection.

In chapter 1, we describe the advancements and challenges for efficient antimicrobial therapy. Even though aPDT is emerging as a potential tool against the worlds tormenting issue of increasing number of drug-resistant pathogens, most PSs used for conventional therapies lack selectivity, target specificity and can undergo selfquenching in biological media due to aggregation. Several strategies to enhance the activity of such PSs have been discussed above which allows for a better solubility and selectivity of the PSs towards the target microbe. This concludes to have a good drug delivery system or chemically altering the molecular skeleton of the PS itself to introduce functionalities to help with problem of quenching and aggregation in aqueous media also aiding a better target selectivity and interaction.

In chapter 2, we describe the conjugating formulation of PSs that have been known in literature for their aPDT potential with an aim to enhance the solubility index of these PSs along with an aim to have better selectivity against the microbes. Hemi-synthesis and synthesis of PSs as polyaminated chlorin-p6 and 5-(4-aminophenyl)-10,15,20tris(4-N-methylpyridinium)porphyrin triiodide is performed and then their water stable and soluble formulation with hyaluronic acid bio-polymer (PS-HA) is achieved.

Structures of porphyrin-HA formulations were characterized by IR, DSC, DLS, zeta