BIMETALLIC RUTHENIUM(II) POLYPYRIDYL COMPLEXES BRIDGED BY A BORON
DIPYRROMETHENE (BODIPY): SYNTHESIS, SPECTROSCOPIC AND PLASMID DNA
PHOTOREACTIONS AND THE IMPACT OF THE 515 NM EFFECT IN
PHOTOSYNTHESIS: MODEL SYSTEM USING β-CAROTENE ACID COMPLEXES
Thesis
Submitted to
The College of Arts and Sciences of the
UNIVERSITY OF DAYTON
In Partial Fulfillment of the Requirements for
The Degree of
Master of Science in Chemistry
By
Ashlee Elizabeth Wertz
Dayton, Ohio
May 2019 BIMETALLIC RUTHENIUM(II) POLYPYRIDYL COMPLEXES BRIDGED BY A BORON
DIPYRROMETHENE (BODIPY): SYNTHESIS, SPECTROSCOPIC AND PLASMID DNA
PHOTOREACTIONS AND THE IMPACT OF THE 515 NM EFFECT IN
PHOTOSYNTHESIS: MODEL SYSTEM USING β-CAROTENE ACID COMPLEXES
Name: Wertz, Ashlee Elizabeth
APPROVED BY:
Shawn M. Swavey, Ph.D., Professor Mann Chair in the Sciences Committee Chair
Mark B. Masthay, Ph.D. Associate Professor. Committee Chair
Jeremy M. Erb, Ph.D. Assistant Professor. Committee Member
ii © Copyright by
Ashlee Elizabeth Wertz
All rights reserved
2019
iii ABSTRACT
BIMETALLIC RUTHENIUM(II) POLYPYRIDYL COMPLEXES BRIDGED BY A BORON
DIPYRROMETHENE (BODIPY): SYNTHESIS, SPECTROSCOPIC AND PLASMID DNA
PHOTOREACTIONS
Name: Wertz, Ashlee Elizabeth University of Dayton
Advisor: Dr. Shawn M. Swavey
Photodynamic therapy (PDT) is a medical technique which utilizes a photosensitizing drug, light of a certain wavelength and molecular oxygen to generate singlet oxygen, a toxic oxidizing
1 species. When present, singlet oxygen ( O2) will rapidly react with surrounding biomolecules, causing cellular damage that ultimately leads to cell death. PDT is an approved medical technique and it has been used for multiple purposes including cases of acne and psoriasis, age-related macular degeneration, and more recently, in the treatment of cancer. To the ends of creating a photosensitizer for PDT, a new pi-extended dipyrrin containing isoquinolpyrrole has been synthesized by solvent free reactions with trifluoroacetic acid (TFA) as a catalyst. The boron- dipyrrin (Bodipy) of the isoquinolpyrrole was synthesized by standard procedures followed by synthesis of the bis-ruthenium(II) Bodipy analog. The spectroscopic properties of this complex show the typical intra-ligand charge transfer transitions (ILCT) along with the Ru(π) to ligand(π*) metal to ligand charge transfer (MLCT) transitions. An intense transition at 608 nm with molar absorptivity greater than 100,000 M-1cm-1 associated with the ππ* transition of the Bodipy core is observed. In acetonitrile solutions the bis-Ru(II)-Bodipy complex generates significant singlet oxygen when irradiated with low energy light. In aqueous solutions the complex is capable of photo-nicking plasmid DNA when irradiated within the photodynamic therapy (PDT) window of
600 to 850 nm.
iv ACKNOWLEDGMENTS
Special thanks are in order to Dr. Shawn M. Swavey who made this thesis possible by allowing me to join his lab and by keeping the project on track to completion. I would like to thank him for the patience he showed in explaining synthesis, electrochemistry and spectroscopy and for always being around to answer my numerous questions. In addition, I am grateful for the time he spent proof reading this thesis and for his valuable feedback.
I would also like to thank the University of Dayton Chemistry Department for funding this work.
Finally, I would like to express my gratitude to my parents, sisters and boyfriend for providing me with support and continuous encouragement throughout my years of study. This accomplishment would not have been possible without them. Thank you.
v TABLE OF CONTENTS: PART 1
ABSTRACT…………………………………………………………………………………….... iv
ACKNOWLEDGMENTS…………………………………………………………………..……..v
LIST OF FIGURES ………………………………………………………………….…………..vii
CHAPTER 1 INTRODUCTION TO PHOTODYNAMIC THERAPY…………………...... 1
Chapter 1 References …………………………………………………………………... 20
CHAPTER 2 EXPERIMENTAL …………………………………………………………..….....29
Materials ……………………………………………………………………………….. 29
Electronic Absorption Spectra ………………………………………………...... 29
Luminescence Spectra ……………………………………………………….………….29
DPBF Studies …………………………………………………………………...... 29
DNA Photocleavage Studies………………… ………………………………………..30
Synthesis ………………………………………………………………………...... 30
Chapter 2 References ……………………………………………………………………32
CHAPTER 3 RESULTS AND DISCUSSION…………………………………………………...33
Synthesis ………………………………………………………………………...... 33
Spectroscopy …………………………………………………………………………….34
DNA Studies …………………………………………………………………………….37
DPBF Studies ……………………………………………………………………………39
Conclusion ………………………………………………………………………………40
Chapter 3 References ……………………………………………………………………40
vi LIST OF FIGURES
Figure 1. Modified Jablonski diagram …………………………………………………………….5
Figure 2. Structure of Porfimer Sodium …………………………………………………………..7
Figure 3. Chemical Structure of Levulan® ………………………………………………………..8
Figure 4. Chemical Structure of Metvixia® ……………………………………………...... 8
Figure 5. Bodipy core which can be extensively modified ………………………………………10
Figure 6. Structure of common Bodipy dyes …………………………………………………….12
Figure 7. Structure of common Bodipy dyes (II) ………………………………………...... 13
Figure 8. Dibromo-aza-Bodipy (ADPM06) …………………………………………...…………13
Figure 9. BF2 -chelated azadipyrromethene dibrominated analogue …………………………….14
Figure 10. Structures of Ru-porphyrin conjugates ……………………………………………….17
2+ Figure 11. [Ru(bpy)2(N–N)] complexes ………………………………………………………..18
Figure 12. Structures of the six different DNA intercalating Ru complexes 18a–f ……... ……...19
Figure 13. Synthetic route for the Bodipy -dye, complex I, and complex II. ……………...…… 34
Figure 14. Absorption spectra of the carbazole Bodipy (blue) and Ru2 carbazole Bodipy (I)
(red) in acetonitrile………………………………………………………………………………..35
Figure 15. UV/vis spectra of complex I (blue) and II (red) in dry acetonitrile at 298 K using a 1 cm quartz cuvette ………………………………………………………………………36
Figure 16. Spectroelectrochemistry of complex I in dry acetonitrile (298 K) with
Et4NPF6 as supporting electrolyte ………………………………………………………………..37
Figure 17. Gel electrophoresis of circular plasmid DNA ………………………………………..38
Figure 18. Gel electrophoresis of circular plasmid DNA (II) ……………………………………39
Figure 19. Time-dependent generation of singlet oxygen upon irradiation at l > 550 nm……….40
vii
CHAPTER 1
INTRODUCTION TO PHOTODYNAMIC THERAPY
Discovery and Background
The history of using light as a therapeutic agent go back many centuries as it had long been used by the Chinese, Egyptians and Indians in the treatment of disease 1. The ancient Greeks developed heliotherapy, a restorative health treatment that involved full body exposure to sunlight. Although the origins of using light as a treatment trace back centuries, it was not until more recently that phototherapy has been widely used in medicine 2. In the eighteenth and nineteenth centuries sunlight was used for the treatment of various conditions such as tuberculosis rickets, scurvy and muscle weakness 3. Phototherapy was developed into a science and popularized by the Danish physician Nils Finsen. Finsen described the successful treatment of smallpox using red light and used ultraviolet light to treat cutaneous tuberculosis. He also initiated the use of carbon arc phototherapy for lupus vulgaris and was awarded the Nobel Prize in 1903 for his work 4,5.
The concept of cell death being induced by the interaction of both light and chemicals has been recognized for over a century. The technique was first reported by medical student Oscar
Raab who observed that Paramecium spp. Protozoans were killed after staining with acridine orange and subsequent exposure to bright light 6. He, along with professor von Tappeiner, demonstrated that acridine exposed to light had a greater effect on Paramecium than either acridine alone, light alone or acridine exposed to light and then added to the paramecium. They had therefore discovered that it was not the light itself, but some product of fluorescence that induced toxicity. Von Tappeiner took over Raab’s research and, with dermatologist Jesionek, published clinical data using eosin as a photosensitizer in the treatment of skin cancer and lupus of the skin. In 1904, von Tappeiner and Jodlbauer reported that the presence of oxygen was a
1 requirement for photosensitizationon 5. In 1907 these experiments were collated into a book in which von Tappeiner coined the term ‘photodynamic therapy’ to describe the phenomenon of oxygen-dependent photosensitization 7.
Uses for Photodynamic Therapy
The field of PDT, a medical technique using light, a photosensitizing drug and molecular oxygen, truly began to form into a practiced medical technique in the 1960’s when Dougherty 9 brought this novel therapy to the attention of a worldwide audience. Skin conditions were among the first types of diseases to be studied for use with PDT. This is due to their easy accessibility to a photosensitizer and light. The Dougherty research group at the Roswell Park Cancer Institute in
Buffalo pioneered skin cancer PDT using the first photosensitizer, a water-soluble mixture of porphyrins that was named ‘haematoporphyrin derivative’ (HpD) and a xenon arc lamp. A more purified preparation of HpD later became known as Photofrin® which will be discussed in detail later. In an early study by Dougherty, 48% of transplanted mouse mammary tumors were cured
10. In 1978, Dougherty reported success in one of the first patient trials with PDT. Twenty-five patients with either primary or secondary skin tumors were treated with HpD followed by exposure to red light from a xenon arc lamp 24–168 h later. All tumors had been resistant to conventional treatment. After PDT ninety-eight lesions completely regressed, 13 exhibited a partial response and only two were resistant to treatment. There were some negative side effects including sunburn, erythema, edema and in some cases skin necrosis, although these effects could be reduced by varying the time interval between drug administration and light exposure. This study demonstrated that PDT could be used successfully in the treatment of various malignant tumors even where conventional therapies had failed 9. Dougherty is referred to as ‘The Father of
PDT’ for bringing PDT to global awareness 10.
Since initial trials with photodynamic therapy there have been many advances in its medical uses. PDT is used to treat neoplastic disease such as actinic keratoses. Actinic keratoses
2 are scaly growths caused by damage from exposure to ultraviolet (UV) radiation. Convincingly,
PDT is an effective treatment for actinic keratoses, with an excellent cosmetic outcome, especially compared with 5-fluorouracil and cryotherapy 11. Other neoplastic diseases that have been demonstrated to show improvements upon treatment with PDT include squamous cell carcinoma, basal cell carcinoma and cutaneous T cell lymphoma 12.
PDT has been used for the treatment of inflammatory diseases such as the most common dermatologic disorder, acne vulgaris. Acne is thought to be primarily caused by the obstruction of sebaceous glands, leading to spread of bacteria, mainly Propionibacterium acnes 13. ALA-PDT
(aminolevulinic acid-PDT) and MAL-PDT (methyl aminolevulinate-PDT) with a light-emitting diode are commonly used as treatments for acne. The efficacy of PDT in acne was first described in a study of 22 patients, where 20% ALA was applied topically to the back with 3-hour occlusion followed by red light irradiation. By the end of the study, there was complete destruction or a 45% decrease in sebaceous gland size 14. PDT has also been demonstrated to have success in the treatment of microbial diseases such as human papillomaviruses (HPV), onychomycosis and cuaneous leishmaniasis 12.
It has been decades since photodynamic therapy was proposed as a useful tool in oncology, but the approach is only beginning to be widely used. PDT drugs have been approved for treatment of carcinoma in situ of the bladder. Its use is not as common as other forms of cancer treatment; however, preliminary data has shown its potential 15. PDT drugs have also been approved for treatment of micro invasive, non-small cell lung cancer in the USA, Japan, and
Europe. Although early disease is often not identified (up to 80% of lung cancer is already sufficiently advanced as to be inoperable at time of diagnosis), detection of disease should improve in the future, when PDT will become an important treatment option 16.
In dermatological oncology, PDT is already a routine treatment. For internal lesions, inexpensive and convenient light sources are now available. However, improved drugs that are
3 more selective and that can be used conveniently and without sustained skin photosensitivity are needed. If such drugs can be developed, then the advantages of PDT could ensure a substantial future role for this type of treatment in oncology 17.
Current state of photosensitizers
PDT uses non-toxic dyes called photosensitizers (PSs) that are activated by the absorption of visible light. The ideal PS structure is very different between anti-cancer drugs and antimicrobial drugs. Most of the PSs used in cancer treatment are based on the tetrapyrrole backbone, a structure similar to that contained in the protoporphyrin prosthetic contained in hemoglobin. Tetrapyrrole backbones occur naturally in several important biomolecules such as heme, chlorophyll and bacteriochlorophyll. Antimicrobial PSs, on the other hand, should have structures that undergo pronounced cationic charges, and in many cases the more charges the better, especially for targeting Gram-negative bacteria 18.
A PS should ideally be a single compound that is stable at room temperature, chemically pure and of known composition. Ideally the compound should have a straightforward synthesis with a relatively high yield. It should have a strong absorption peak in the red to near-infrared spectral region (between 600 and 800 nm) because absorption of single photons with wavelengths longer than 800 nm does not provide enough energy to excite oxygen to its singlet state and this longer wavelength light can be absorbed by molecules of H2O in the body. However, penetration of light into tissue increases with wavelength so agents with strong absorbance in the deep-red spectral region tend to make much more efficient PSs 19. PSs should possess a substantial triplet quantum yield leading to good production of reactive oxygen species upon irradiation. It should have retention by target tissue but be rapidly excreted from normal tissue, inducing a low systemic toxicity 19.
4
The most effective PSs tend to be relatively hydrophobic compounds that rapidly diffuse into tumor cells and localize in intracellular membrane structures such as the mitochondria and endoplasmic reticulum. More polar compounds tend to be taken up by the active process of adsorptive or fluid phase endocytosis, and this process is slower than passive diffusion, necessitating a longer drug light interval20.
Mechanism of Action
The basis of PDT is the initiation of photochemistry at target sites. The two main steps in the process of PDT are the injection of a photosensitizer and subsequent light irradiation at a specific wavelength that is appropriate for absorption by the sensitizer 21. The photophysical processes involved in PDT are shown in the figure below.
Figure 1: Modified Jablonski diagram. Photophysical processes involved in photodynamic therapy 22. 1.) Light energy is absorbed by the photosensitizer which is transformed from its singlet ground state (S0) to the short-lived first excited singlet-state (S1). 2) The S1 PS can return to the
S0-state by emitting the absorbed energy as fluorescence or 3) by internal conversion. 4)
Alternatively, it can also convert to the relatively long-lived first excited triplet state (T1) by intersystem crossing. 5) The T1 can return to the S0-state by emitting phosphorescence.
5
Alternatively, T1 may initiate photochemical reactions directly by generating reactive, cytotoxic
3 free radicals, or indirectly by transferring its energy to ground state oxygen ( O2). This step leads
1 to the formation of excited state oxygen, singlet oxygen ( O2), which causes photo-oxidative damage to cellular targets 22. There are two proposed types of photochemical pathways, known as type I and type II reactions, both of which necessarily require the availability of oxygen. 6) In the type I reaction, the PS interacts with a biomolecule (or oxygen) resulting in hydrogen atom (or electron) transfer that leads to the production of free radicals. 7)In the type II reaction, energy is transferred to molecular oxygen and singlet oxygen is generated 23. Both reaction types can occur either simultaneously or exclusively, depending on the chemical structure of the photosensitizer, however, the type II mechanism is generally believed to be the most common 24.
1 The lifetime of singlet oxygen ( O2) is very short (~10–320 ns), limiting its diffusion to only approximately 10–55 nm in cells 24. Thus, photodynamic damage is likely to occur only in the immediate vicinity of the photosensitizer 25. It is thought that the localization of the PS in different organelles plays a major role in the type of cell death mechanism that dominates, but other factors such as the overall PDT dose also plays a major role. Overall, it is accepted that apoptosis is the principal modality of cell death when cells are treated with PDT in vitro 19.
Currently Approved Photosensitizers
HpD and Photofrin were the first PSs to receive regulatory approval, and these and similar preparations are still in widespread use around the world 18. Porfimer sodium
(Photofrin®) was the PS that brought PDT to a worldwide audience 21. This photosensitizer is a proprietary combination of monomers, dimers and oligomers, linked by esters and ethers with all components being required for chemical activity 22.
6
Figure 2: Structure of Porfimer Sodium 26 the first photosensitizer to receive FDA approval.
In the US, Photofrin® is FDA approved for early and late endobronchial lesions as well as Barrett’s esophagus and esophagealobstructing lesions 27. Off label use has been extensive as well. The drug is approved worldwide for a number of additional diseases including cancers of the esophagus, lung and bladder. While Photofrin® has shown positive results and promising possibilities there are also downfalls to its use. Porfimer sodium is activated by red light at 630 nm. Photons of this wavelength do not penetrate below a few millimeters, therefore, this drug is only suitable for superficial tumors or ones that can be reached via fiber optic procedures 28. The drug is not highly selective at 2 mg/kg and there is significant prolonged photosensitivity. Most patients that have received treatment are required to stay out of sunlight for at least 4 weeks.
While the drug does appear to concentrate in the tissue being treated, the normal tissue also reacts, just less intensely. This can manifest as swelling of the skin for cutaneous lesions 29.
Another drawback of Photofrin® is that it received FDA approval as a complex mixture of oligomeric compounds. It is advantageous to have a drug be a pure, known compound.
There are two other FDA approved photosensitizers on the market. The first is Levulan®
(aminolevulinic acid HCl)
7
Figure 3: Chemical Structure of Levulan® a photosensitizer used in the treatment of actinic keratosis This photosensitizer is used for the treatment of minimally to moderately thick actinic keratosis of the face or scalp. This drug is activated by blue, rather than red light 30. The other is
Metvixia® which is also used to treat actinic keratosis and uses a red light of 570 to 670 nm 31.
Figure 4: Chemical Structure of Metvixia® an FDA approved photosensitizer used in the treatment of actinic keratosis.
Current Research in Photosensitizers
To overcome some of the drawbacks currently associated with PDT and improve the treatment efficacy, several strategies have been employed for the development of more tumor- selective agents with reduced side effects, especially skin phototoxicity. The bulk of research into new PSs has focused on porphyrin-type, chlorin-type, phthalocyanine-type and Bodipy-type 32.
The Bodipy type PS will be discussed in detail in the following section. The porhyrin-type PSs were mainly developed and clinically used in the last few decades. Side-chains containing function groups such as nitrogen atoms 33, carboxylic acid 34, and sugar 35 are frequently incorporated into the porphyrin skeleton. For example, a novel porphyrin-based PS (5,10,15,20- tetrakis[(5-diethylamino)pentyl] porphyrin, TDPP) with four diethylaminopentyl side-chains
33 1 recently reported by Li et al. showed a high O2 yield with the ability to kill human esophageal cancer cell lines (Eca-109) and significantly reduce the growth of Eca-109 xenograft tumors in
8
BABL/c nude mice. Chlorin-type PSs attract considerable attention due to their intense absorption in the relatively harmless NIR region, which can penetrate deeply into biological tissues. However, the development of chlorin derivatives was significantly limited by their poor water solubility. Therefore, chlorin-type PSs have been modified by conjugation with amino acids, peptides, and sugars to improve their solubility for PDT investigations. For example, Meng et al.36 prepared a series of chlorin P6-based water-soluble amino acid conjugates.
Phthalocyanine-type PSs were thought to be most promising, however, the low solubility and π-π stacking in these molecules limited their further clinical application. Strategies to overcome these disadvantages can involve incorporations of cationic 37 or anionic groups 38, peptides 39, β- cyclodextrins 40, crown ethers 41, glycerinum 42 and so on. Despite the extensive research performed to develop new and improved PS, only a few second-generation PSs, such as
MetVix®, Photochlor® and NPe6, have been approved for the clinical treatment of cancer 22.
The discovery of novel PS molecules with desired pharmaceutical properties and the application of novel PS in clinical trials are challenging tasks. During the last several years, most research work is based on the modification and optimization of old-style PSs. The most recent activity in the PS field for PDT of cancer has been considerable, and the design of non-porphyrin PSs, which possess shorter periods of photosensitization, longer activation wavelengths and higher singlet oxygen yield, still attracts more attention in the field of anticancer PDT 32.
Boron Dipyrromethanes (Bodipy)
A new class of PDT agents has emerged that are based on the 4,4-difluoro-4-bora-3a,4a- diaza-s-indacene (Bodipy) core. Bodipy’s have many ideal photosensitizer characteristics including a large molar absorptive coefficient, high chemical stability, photostability 43, and high light-dark toxicity ratios.44
The aza-Bodipy was first synthesized by Rogers in 1943 as a coloring agent 45 and in
1968 work by Treibs and Kruezer discovered the famous 4,4-difluoro-4-bora-3a,4a-diaza-s-
9 indacene Bodipy. Bodipy derivatives can be extensively modified around the core. The absorption- and fluorescence-spectroscopic properties of members of the Bodipy family are highly influenced by these modifications and the extent of electron delocalization around the central framework 46.
Figure 5: Bodipy core which can be extensively modified.
Applications for Bodipy Dyes
Numerous useful applications have been discovered for Bodipy dyes outside of PDT. In
2006, some 729 patents and 1074 journal articles were published that described the multifarious applications of these dyes and many advances have been made since. One application is for use in biological labeling. The use of Bodipy as an effective biological label has been complemented by its known propensity to function as a tunable laser dye 47. Bodipy dyes have also been used as both an oxidative and reductive sensitizer due to the main structural framework undergoing reversible oxidation and reduction processes at accessible potentials 48. There is high potential for the use of Bodipy dyes as chemical sensors that operate by fluorescence modulation. Daub and
Rurack 49 were the first to show the potential for Bodipy dyes in this field, and their original research has been followed by countless examples of Bodipy -based fluorescent molecular sensors. The excellent thermal and photochemical stability, high fluorescence quantum yield, negligible triplet-state formation, intense absorption profile, and chemical robustness have all
10 added to the general attractiveness of these materials 50. The good solubility of these dyes in most common solvents (excluding water) should also be noted.
Bodipy Dyes as Photosensitizers
The electronic-rich property possessed by the Bodipy core makes it useful for chemical modification, especially the electrophilic aromatic substitution with halogens. This is beneficial because in order to be useful in PDT Bodipy PSs are often equipped with heavy halogen atoms, such as Br and I. Without this modification Bodipy dyes are excited into high level singlet states, however photo-damage in PDT is thought to occur predominantly via triplet excited states.
Adding a heavy atom promotes efficient triplet state population 28.
“Tetramethyl- Bodipy” (Figure 6A), which does not contain a halogen does not significantly populate triple states on excitation and has a poor quantum yield for singlet oxygen generation. The compound in figure 6B which is a diiodo-analog was investigated. Measurements
1 of rate and quantum yield for O2 generation using 1,3-diphenylisobenzofuran revealed high efficiencies for this process in both polar and non-polar solvents 51. This compound was shown to have high light-to-dark photocytotoxicity ratios and high oxidation potentials that may protect
Bodipy from self-oxidation. In a study by Lim it was found that iodination of meso-aryl substituents has less impact than of α or β attached iodines. However, incorporation of a meso-
1 ethylene carboxylic acid group as in Figure 6C improved the rate of O2 generation and light- induced photocytoxicities 44. Additional studies have shown that introduction of iondines at the
3- and 5- positions increases fluorescence 52.
11
A B C
1 Figure 6: Structure of common Bodipy dyes: A) “Tetramethyl-Bodipy” O2 generation rel. rate 1 0.48 (methylene blue) B) “Tetramethyl-Bodipy with core attached iodines” O2 generation rel. rate 23.9 (methylene blue) C) “Tetramethyl-Bodipy with core attached iodines and meso-ethylene 1 carboxylic acid group” O2 generation rel. rate 24.6 (methylene blue).
An important property of PDT Bodipy dyes is to have high photocytotoxicity in vitro when light is present, but low activity when there is no light irradiation. In was shown that compounds which have iodine atoms directly attached to the Bodipy core show the highest photocytotoxic activity when exposed to light. Meso-substiution with a para-iodoaryl group did not alter the photocytoxicity significantly (Figure 7A) however, further substitution with iodine atoms on the two pyrrolic 4-carbon to yield ether compounds (Figure 7B,C) improved the activity by up to 100-fold. This demonstrates the importance of having halogen atoms directly substituted on the Bodipy pyrollic carbon-4 position in accordance with the high photocytotoxic rates when exposed to light irradiation 53.
12
A B C
Figure 7: Structure of common Bodipy Dyes (II): A) “Tetramethyl-Bodipy with meso substituted para-iodoaryl group” B) “Tetramethyl-Bodipy with core attached iodines and para-iodaryl group” C) “Tetramethyl-Bodipy with core attached iodines.” Cell death in PDT can be reduced under depleted oxygen levels (e.g. hypoxic in cancer cells), therefore it is useful for a compound to retain significant activity under these conditions.
This property is retained in the compound shown below which has the modification of the meso- carbon substituted by nitrogen.
Figure 8: Dibromo-aza-Bodipy (ADPM06). Activity is retained even under depleted oxygen levels.
Apoptosis is initiated in PDT by the compound shown in Figure 8 as a result of active oxygen species generated around the ER. This is accomplished by activation of several inhibitor and executioner caspases 54. In addition to showing activity under hypoxic conditions,
13 bromination of aza- Bodipy 2,6- positions results in at least a four-fold reduction in fluorescence
QY and an increased population of triplet states upon excitation 55.
Some PDT side-effects may arise from prolonged light sensitivity. Bodipy dyes can be used to selectively quench intersystem crossing by photoinduced electron transfer and minimize damage to healthy tissues. Aza- Bodipy dyes with a non-conjugated but proximal amine may undergo rapid relaxation via photoinduced electron transfer when the amine is not protonated.
However, a larger portion of the amine would be protonated in the relatively acidic interstitial fluid that surrounds tumors. Photoinduced electron transfer would selectively diminish in those regions and the cytoxic effect would be greater around cancerous cells than healthy ones 56. The compound in Figure 9 was shown to generate more singlet oxygen in acidic than neutral media, however, photocytoxicities of this compound in vivo have not yet been compared with closely related compounds that lack the amine group. Studies have been promising, but clinical potential is still an open question.
Figure 9: BF2 -chelated azadipyrromethene dibrominated analogue: generates more singlet oxygen in acidic rather than neutral media.
Since the early publication by O'Shea et al. of aza- Bodipy PSs in 2002, the Bodipy class
PSs have shown great promise in a very short time. Bodipy dyes show many advantages over other agents: first, they usually have a large molar absorptive coefficient; second, they have
14 extremely high chemical stability and photostability; third, they have facile availability and can be easily structurally modified to modulate their properties. In addition, the recent explosion in knowledge about Bodipy’s as fluorescence dyes, simple methods for converting from fluorescence dyes to PSs and relatively reliable theoretical tools could provide ideas for designing new Bodipy -based PSs 57. One downfall is that the intrinsic absorption maxima of simple Bodipy dyes (510 – 530 nm) is shorter than ideal 29. Other cons of using Bodipy dyes in PDT is that they do not have a high solubility in water and because of the very short lifetime of the excited state they fluoresce but do not generate a large singlet oxygen amount. Modifications must be made to the core Bodipy structure in order to improve these properties and described above are only a few of the modifications that have been studied to date.
Ruthenium (II) Complexes in PDT
Ru(II) complexes also show high potential as PSs in PDT. There are many properties that make ruthenium compounds well suited to medicinal applications. The first is the rate of ligand exchange. Ligand exchange is an important determinant of biological activity as very few metal drugs reach the biological target without being modified. Ru complexes are considered prodrugs meaning the compound that is administered to the patient is not the active form 58. However, because Ru complexes can access a range of oxidation states, a second beneficial feature, Ru(III) complexes are reduced into a more active Ru(II) form when localized in a hypoxic environment, which is a property characteristic of tumors 59. Oxidation states Ru(II) to Ru(IV) can be accessed under physiological conditions 60 and the redox potential of a complex can be modified by varying the ligands to improve the effectiveness of a drug in vivo. Ruthenium is also useful in medicine because of the ability of ruthenium to mimic iron binding to certain biological molecules. Rapidly diving cells have greater requirement for iron, therefore, they increase the number of transferrin receptors located on their cell surfaces. This facilitates the transportation of
Ru into tumor cells 61. Other features of Ru complexes include their general lower systems
15 toxicity compared to platinum complexes and the ability of these complexes to have different geometries allowing for the design of compounds with a specific cellular target 58. A number of
Ru complexes have previously been shown to display promising anticancer activities, and two of them, NAMI-A and KP109, have entered clinical trials. 62,63
The use of prodrugs such as Ru(II) complexes is an appealing way to reduce the system toxicity of a drug candidate 64. In order to activate the prodrug two different types of stimuli can be employed, either an internal stimuli (hypoxia, cellular conditions) or an external stimuli
(temperature, light). The use of an external stimuli is more advantageous because it gives physicians greater spatial and temporal control over the generation of the toxic molecule, whereas, when an internal stimuli is relied upon one is completely reliant upon intracellular parameters. As of today, the most common applied technique to induce the formation of active species is via light irradiation 65,66.
Ruthenium-containing porphyrin PSs.
The derivation of the porphyrin core with metal PSs is an appealing opportunity to improve the activity of a PS. This functionalization was exploited for the first time by Brunner and coworkers 67,68. They synthesized hemotoporphyrin-platinum conjugates to combine the strong anti-cancer activity of platinum based drugs with the phototoxic effect of porphyrins. The metal derivatization of a porhyrin core can enhance the intrinsic properties of a PS by modifying its physio-chemical characteristics. For example, the metal fragment can change the lipophilicity of the PS, increase its water solubility or improve its cellular uptake.
Several research groups have recently evaluated the possibility of introducing Ru(II) moieties on the periphery of porphyrins. For example, Therrien et al. synthesized a wide range of
Ru-modified porphyrin systems and studied their biological performances 69. They appended a number of Ru-arene fragments to the meso-4’-tetrapyridylporphyrin scaffold to evaluate the
16 influence of the different aromatic moieties (Figure 10). All the compounds were found to induce
60-80% morality in human Me300 melanoma cells at 10 µM concentration, using light at 652 nm with a dose of 5 J cm-2. The photoactivity of the functionalized system was found to be independent of the nature of the arene. This flexibility can give access to the use of arenes which are derivatized with targeting agents or chemo-therapeutic compounds.
Figure 10: Structures of Ru-porphyrin conjugates 69
Ruthenium Polypyridyl Complexes
Beginning in the 1950s, Dwyer et al. published the first articles on the biological activity of bipyridine, phenanthroline, and terpyridine complexes, their in vivo toxicity and anticancer properties as well as for their antibacterial and enzyme-inhibition activity 70. Despite early results, the complexes have only recently received attention as potential anticancer agents. Metal polypyridyl complexes are characterized by high structural versatility due to the different coordination modes of transition metals and to the wide range of ligands commercially and synthetically accessible. Such flexibility can be exploited to obtain a variety of shapes and
17 physicochemical properties (e.g. hydrophobicity) and reactivities, which can be tuned to improve the effectiveness of this class of compounds in chemotherapy.
Complexes such as cis-[Ru(bpy)2Cl2], mer-[Ru(terpy)- (bpy)Cl], and mer-[Ru(terpy)Cl3]
(where terpy = 2,2:6,2-terpyridine) were among the earliest Ru polypyridyl complexes to be tested for anticancer activity 71. In HeLa and L1210 cells as well as in vivo, the latter complex was found to be more cytotoxic than the other two derivatives, which is consistent with its ability to form intrastrand DNA cross-links. More recently, the cytotoxicity and metabolic effects of
2+ 72 related [Ru(bpy)2(N–N)] complexes (Figure 11) were studied by Schatzschneider, Ott et al. .
The dppn derivative 14 shows good antiproliferative properties against the HT-29 and MCF-7 cell lines. The complex is as cytotoxic as cisplatin for these cell lines, having IC50 values of 6.4
μm for HT-29 and 3.3 μm for MCF-7 cells.
2 Figure 11: The cytotoxicity and metabolic effects of these [Ru(bpy)2(N–N)] + complexes were studied. 14 showed good antiproliferative properties against the HT-29 and MCF-7 cell lines.
The application of ruthenium complexes as PSs is a reasonable approach due to their
2+ tunable photophysics. This was shown by synthesizing six [Ru(bipy)2dppz] complexes with different functional groups on the dppz ligand. 73
18
Figure 12: Structures of the six different DNA intercalating Ru complexes 18a–f 73
The presence of the dppz intercalative ligand was meant to increase the affinity of the compounds for DNA, so that a targeted delivery of singlet oxygen to the genetic material can be achieved. All Ru complexes were found to be non-toxic (up to 100 mM) to both normal fetal lung problast cells and cervical cancer HeLa cells in the dark. Nevertheless, the amino- and methoxy- substituted Ru complexes showed impressive photoactivities. When HeLa cells were irradiated
-2 with a light dose of 9.27 J cm at 420 nm, IC50 values in the low micromolar range were obtained for a and b. A and b also showed good efficiency in generating strand breaks of supercoiled plasmid DNA upon light irradiation. This feature strongly suggested the involvement of DNA in the mechanism of phototoxicity 71.
2+ Properties of Ru(bpy)3
The conventional Ru(II) polyimine complexes show weak absorption in the visible region
(ɛ ˂ 20,000 M-1 cm-1) in the region beyond 400 nm, and the absorption maxima are usually at 450 nm. Furthermore, typically the triplet excited state lifetimes of the typical Ru(II) complexes are usually shorter than 5 µs 74, 75, 76, 77, 78,. These photophysical features are detrimental to the applications of Ru(II) polyimine complexes as triplet photosensitizers. To improve these features and maintain the integrity of the properties displayed by the individual components, recent work in this area has focused on tethering ruthenium (II) complexes to the meso-positions of the
19
Bodipy dyes. By extending the distance between the Bodipy core and the Ru(II) polypyridyl complexes, while maintaining a covalent connection, the hope has been to limit the electronic communication between the emitting centers and thus generate dual emission 79.
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28
CHAPTER 2
EXPERIMENTAL
Materials
All chemicals were reagent grade and used without further purification. Isoquino[5,6- c]pyrrole,1 and cis-dichloro(bis-2,2'-bipyridine) ruthenium(II) 2 were synthesized as previously described. Chromatography was performed on a Teledyne CombiflashRf+ equipped with UV detection. Elemental analysis was performed at Atlantic Microlabs Inc. Norcross, Georgia. 1H
NMR spectra were recorded on a Bruker 300 MHz NMR spectrophotomer at 298 K.
Electronic absorption spectra
Electronic absorption spectra were recorded at room temperature using an HP8453 photodiode array spectrophotometer with 2 nm resolution. All spectra were recorded at 298 K.
Luminescence Spectra
Room temperature luminescence spectra in a 1 cm quartz spectrophotometer fluorescence cell (Starna) in DCM were run on a Cary Eclipse fluorescence spectrophotometer.
DPBF Studies
Acetonitrile solutions of 1,3-diphenylisobenzofuran (DPBF, singlet oxygen quencher) and the complexes at roughly a 20 to 1 ratio in quartz cuvettes were irradiated in the presence of oxygen using a 300 W mercury-arc lamp equipped with a long band pass filter cutting off wavelengths below 550 nm. The progress of singlet oxygen production, monitored using the
HP8453 photodiode array spectrometer, was determined by observing the decrease in the maximum absorption band at 411 nm associated with the singlet oxygen trap DPBF as a function of irradiation time.
29 DNA photocleavage studies
Gel electrophoresis was performed using premade E-GelEx 1% agarose gels and run using an Invitrogen E-Gel power snap system (Thermo Fisher). Circular plasmid DNA (pUC18,
Bayou Biolabs) was used. Samples were irradiated with a 300 W mercury arc lamp equipped with a long band pass filter to block out wavelengths less than 550 nm.
Synthesis
Isoquinol-carbazole boron dipyrromethene
In a 100 mL round bottom flask 0.070 g (0.42 mmoles) of isoquino[5,6-c]pyrrole and
0.14 g (0.62 mmoles) of 9-ethyl-3-carbazolecarboxaldehyde (Sigma-Aldrich) in ca. 5 mL of
DCM: methanol (4:1). To this mixture was added ca. 100 µL of trifluoroacetic acid (Aldrich) followed by removal of the solvent under reduced pressure. The resultant homogenous solid was heated in a water bath at 70 °C for 10 minutes. The bright green/blue paste dissolved in a minimum of DCM and neutralized with triethylamine until the solution turned a bright red/purple.
The reaction mixture was purified by column chromatography on silica gel collecting a bright red/purple band at 0.5% methanol in DCM. The isolated dipyrrin was then reacted with boron trifluoroetherate by a standard procedure. Under nitrogen atmosphere in a 25 mL round bottom flask 3 mL of dry DCM was added to 0.080 g of isoquinol-carbazole dipyrrin. To this solution, triethylamine (100 µL) was added and stirred for 5 minutes at room temperature, at which time,
(150 µL) boron trifluoroetherate was added, and the reaction mixture was allowed to stir overnight under nitrogen at room temperature. The reaction mixture was washed with distilled water, extracted into dichloromethane and the solvent was removed. The reaction mixture was purified by column chromatography with 0.5% methanol in DCM and recrystallized from a
DCM/hexanes mixture to give a dark blue powder, 0.062 g (19% yield). 1H NMR (300 MHz,
CDCl3) δ 8.86 (s, 1H), 8.43 (s, 1H), 8.14 (d, J = 7.8 Hz, 2H), 8.01 (d, J = 8.4 Hz, 2H), 7.81 (d, J =
30 8.3 Hz, 1H), 7.72 (d, J = 8.6 Hz, 1H), 7.63 (d, J = 3.7 Hz, 2H), 7.38 (ddd, J = 14.8, 13.0, 7.8 Hz,
10H), 7.16 (t, J = 7.2 Hz, 3H), 6.42 (d, J = 8.5 Hz, 1H), 6.31 (d, J = 8.4 Hz, 1H), 5.37 (s, 2H),
4.62 (d, J = 7.2 Hz, 2H), 4.43 – 4.22 (m, 2H), 1.68 (t, J = 7.1 Hz, 3H), 1.40 (t, J = 7.1 Hz, 3H).
Anal. Calcd. for C52H37N12N6F2B •0.33 CH2Cl2 C, 76.37; H, 4.61; N, 10.21; F, 4.62. Found; C,
76.69; H, 4.93, N; 10.22; F, 4.86.
Bis-Ru(bpy)2Cl-Isoquinol-carbazole-BDP (I)
Under a nitrogen atmosphere 0.014 g (0.018 mmoles) of the BDP-dye and 0.019 g (0.040 mmoles) cis-Ru(bpy)2Cl2 were refluxed for 2 h in 5 mL absolute ethanol. After cooling to room temperature, the mixture was added dropwise to 50 mL of an aqueous NH4PF6 solution. The precipitate was filtered, washed with D.I. water and air dried. The green/blue powder was taken up in a minimum (2 mL) of acetonitrile and added dropwise to 50 mL of diethylether followed by filtration and then air dried to give 0.028 g (79% yield) of product. Anal. Calcd. for
C92H69N14BCl2P2F14Ru2 •6H2O C, 52.86; H, 3.91; N, 9.38; F, 12.72. Found; C, 52.43; H, 3.65, N;
9.05; F, 12.53.
Bis-Ru(bpy)2Cl-Isoquinol-carbazole (II)
In 8.0 mL of absolute ethanol under nitrogen atmosphere 0.030 g (0.040 mmoles) of
Isoquinol-carbazole dipyrrin and 0.043 g (0.089 mmoles) of cis-Ru(bpy)2Cl2 were refluxed for 4 h. The reaction mixture was cooled to room temperature and precipitated by addition to 50 mL of aqueous NH4PF6. The precipitate was filtered and washed several times with D.I. water. The brown powder was taken up in a minimum of acetonitrile (2 mL) and added to 50 mL of diethylether. The precipitate was filtered and air dried to give 0.72 g (93% yield). (Yield 24 mg,
14%). Anal. Calcd. for C92H70N14Cl2P2F14Ru2 •2H2O C, 56.07; H, 3.78; N, 9.95; F, 11.57. Found;
C, 56.16; H, 3.76, N; 9.93; F, 11.94.
31
Chapter 2 References
1. Lash, T.D. and Virajkumar, G. Porphyrins with Exocyclic Rings 15. Synthesis of Quino- and
Isoquinoporphyrins, Aza Analogues of the Naphthoporphyrins. J. Org. Chem. 2000, 65, 8020-
8026.
2. Sullivan, B.P.; Salmon, D.J.; Meyer, T.J. Mixed phosphine 2,2’-bipyridine complexes of ruthenium. Inorg.Chem. 1978, 17, 3334-3341.
32 CHAPTER 3
RESULTS AND DISCUSSION
Synthesis
For the past few years Dr. Swavey’s laboratory has been interested in developing a bonding motif capable of accommodating a strong orbital interaction between ruthenium(II) polypyridyl complexes and 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (Bodipy) dyes. To this end, his group has recently described the synthesis of three π-extended isoquinol based Bodipy
+ dyes as a bridging ligand coordinated to two {Ru(II)(bpy)2Cl} moieties. The aldehydes utilized in the synthesis of the dipyrrin Bodipy -precursors were chosen to offer little in the way of steric interference; thereby, allowing for acceptable yields. The synthetic route to these dipyrrins involved heating the appropriate aldehyde with isoquinolpyrrole, in the absence of solvent, followed by chromatography. Temperatures needed for these reactions were manageable using only a water bath. To expand this work, we have sought to determine if this technique is applicable to bulkier more sterically hindered aldehydes, e.g. pentafluorobenzaldehyde, 9-ethyl-3- carbazolecarboxaldehyde, 1-pyrenecarboxaldehyde, and 4-(diphenylamino)benzaldehyde.
Heating mixtures of these aldehydes and the isoquinolpyrrole in a water bath was insufficient to generate the products desired, leading us to use oil baths at temperatures ranging from 180-190
°C. Unfortunately, these temperatures resulted not only in the desired dipyrrins but also numerous unidentified byproducts making it difficult to purify. To overcome the high temperatures trifluoroacetic acid (TFA) was added to a mixture of the bulky aldehyde, 9- ethylcarbazolecarboxaldehyde, and isoquinolpyrrole. The reaction mixture was heated to 80 °C in a water bath generating a bright green paste. The paste was dissolved in dichloromethane and neutralized with triethylamine to give a violet solution which was chromatographed on silica using 0.5% methanol/dichloromethane as eluent, scheme 1. The Bodipy dye was synthesized by a
33 standard procedure; once isolated it was reacted with a slight excess of cis-Ru(bpy)2Cl2 to give the desired complex I. For comparison, the dipyrrin was reacted with Ru(bpy)2Cl2 leading to complex II in good yields, scheme 1.
Figure 13: Synthetic route for the Bodipy -dye, complex I, and complex II.
9-ethyl-3-carbazolecarboxaldehyde and 4-(diphenylamino)benzaldehyde were the bulky aldehydes that were chosen and products were attempted to be synthesized as shown in scheme 1.
The synthesis with 4-(diphenylamino) benzaldehyde was not successful and the product was not able to be fully characterized.
34
Spectroscopy
Absorption spectra of the Bodipy and Ru2-Bodipy complexes were performed in acetonitrile (Figure 1). The carbazole Bodipy compound showed an intense absorption band in the visible region at approximately 580 nm associated with the π-π* transition of the dipyrrin.
The Ru2-Bodipy complex (I) displays the traditional high energy Bodipy π-π* transitions near
288 nm with lower energy Ru(dπ) to bpy (π*) MLCT transitions near 350 nm and Ru (dπ) to pyrin (π*) transitions at approximately 500 nm. It was observed that the Bodipy π-π* transition shifts 25-30 nm, from approximately 580 nm to 610 nm. This red shift can be explained by the extension of the pi system once cis-Ru(bpy)2Cl2 is coordinated.
1.2
1.0
0.8
0.6
Absorbance 0.4
0.2
0.0 300 400 500 600 700 800 , nm
Figure 14: Absorption spectra of the carbazole Bodipy (blue) and Ru2 carbazole Bodipy (I) (red) in acetonitrile.
The electronic absorption and emission spectra of complexes I and II, as well as the
Bodipy -dye scheme 1, were determined in dry acetonitrile using a 1 cm quartz cuvette.
Comparison of complexes I (blue) and II (red) are illustrated in Fig. 2. Both complexes show an intense absorption at 288 nm associated with the bipyridyl (π-π*) intraligand charge transfer
(ILCT) transition. A Ru(dπ) to bpy(π*) metal to ligand charge transfer (MLCT) band appears at
345 nm for II and is red shifted to 379 nm upon coordination to BF2, I (blue Fig. 2). The most
35 striking difference for I and II appears in the visible region of the spectrum where II shows a very broad absorption from 450- 600 nm representing overlapping Ru(dπ) to dipyrrin(π*) and ππ* dipyrrin transitions. In contrast, upon coordination of BF2, leading to alignment of the pyrrole units, an intense absorption due primarily to the ππ* BDP transition 1, 2 appears at 608 nm with a higher energy shoulder associated with the Ru(dπ) to dipyrrin(π*) MLCT band. The absorption observed at 608 nm (I, blue) is red shifted by ca. 30 nm compared to the peak absorption of the
+ BDP-dye strongly suggesting that coordination of the {Ru(bpy)2Cl} moieties alters the orbital energies of the dipyrrin core.
1.4e+5
1.2e+5
1.0e+5
-1
cm 8.0e+4
-1
, M 6.0e+4
4.0e+4
2.0e+4
0.0 300 400 500 600 700 800
, nm
Figure 15: UV/vis spectra of complex I (blue) and II (red) in dry acetonitrile at 298 K using a 1 cm quartz cuvette.
To support the spectroscopic assignments spectroelectrochemical studies were performed on solutions of I by monitoring spectroscopic changes after oxidation of the Ru(II) centers to
Ru(III). The results of this experiment, Fig. 3, show that after oxidation, the MLCT transitions disappear and the ππ* dipyrrin ILCT band is blue shifted to 588 nm (Fig. 3 red).
36
0.30
0.25
0.20
0.15
Absorbance 0.10
0.05
0.00 300 400 500 600 700
, nm
Figure 16: Spectroelectrochemistry of complex I in dry acetonitrile (298 K) with Et4NPF6 as supporting electrolyte, high area platinum mesh working electrode, platinum wire auxiliary, and a Ag/AgCl reference electrode. Initial scan (blue), after oxidation at 1.00 V (red).
Upon reduction of the Ru(III) centers back to Ru(II) 95% of the original spectrum is obtained, indicating that the complex is intact throughout the experiment. It should be noted that attempts to measure the luminescence of I revealed that the emission from the BDP-core is most likely quenched by the Ru(II) moieties, similar to previous studies. 3, 4
DNA Studies
As mentioned in the introduction, one potential application of the Ru(II)2-Bodipy-bridged complex is in the field of phototherapeutics. Typically, the photosensitizer is activated by light to generate an excited state capable of transferring energy or electrons to cellular molecules generating localized reactive radicals or singlet oxygen capable of destroying cellular function.5
One biological target for phototherapeutics is cellular DNA. To this end, measuring the ability of the photosensitizer to cleave or nick plasmid DNA when irradiated within the photodynamic window is the typical starting point for evaluating photosensitizers. Aqueous solutions of circular
37 plasmid DNA and complex I at a ratio of 5/1 base-pairs (bp)/complex were irradiated with a 300
W mercury arc lamp equipped with a filter to block out wavelengths less than 550 nm. Using this filter ensures that only light of energy within the PDT window irradiate the samples.
1 2 3 4 5 6 7 8 9 10
Figure 17: Gel electrophoresis of circular plasmid DNA (pUC18) in the absence (lane 1) and presence (lanes 2-10) of complex I at a 5:1 base pair to complex ratio. Samples were irradiated with a 300 W mercury arc lamp equipped with a long pass filter, cutting off wavelengths below 550 nm. Samples were taken at 10 minute intervals.
Aliquots were removed at 10-minute intervals and gel electrophoresis was run to determine the ability of the complexes to photo-react with the circular plasmid DNA. The highly negative charge of the phosphate backbone of circular plasmid DNA causes rapid migration through the gel. Results of this study are illustrated in Fig. 4 (complex I) with lane 1 being plasmid DNA in the absence of complex and irradiation. Lane 2 (Fig. 4) represents plasmid DNA in the presence of the respective complex in the absence of irradiation. After 80 minutes of irradiation I shows significant DNA-nicking (Fig. 4), in addition, migration of DNA illustrated in lanes 5-10 Fig. 4 shows light induced retarded migration of the un-nicked DNA.
Complex I was also tested for its ability to cleave DNA using a 420 nm filter. A 5:1 base pair to complex ratio was used and samples were taken at 4-minute intervals (Figure 5). Lane 1, being plasmid DNA in the absence of complex and irradiation, was problematic and did not show up clearly on the gel (perhaps due to inadequate centrifugation). Despite this, nicking can still clearly be seen in the other lanes
38
1 2 3 4 5 6 7 8 9 10
Figure 18: Gel electrophoresis of circular plasmid DNA (pUC18) in the absence (lane 1) and presence (lanes 2-10) of complex I at a 5:1 base pair to complex ratio. Samples were irradiated with a 300 W mercury arc lamp equipped with a long pass filter, cutting off wavelengths below 420 nm. Samples were taken at 4-minute intervals.
Complex I has significant absorption within the 600-850 nm region. The proximity of the photosensitizer to biomolecules like DNA plays an important role especially if generating singlet oxygen is the mechanism of the phototherapeutic agent. Since the lifetime of singlet oxygen is as short as 3 ms; therefore, if the photosensitizer is electrostatically bound to the DNA then any singlet oxygen generated will have time to react with the DNA.
DPBF Studies
Acetonitrile solutions of complex I and the singlet oxygen trap 6 1,3- diphenylisobenzofuran (DPBF) were irradiated with a 300 W mercury arc lamp equipped with a
550 nm band pass filter. The production of singlet oxygen was monitored spectrophotometrically as the absorption of DPBF (lmax = 411 nm) decreases upon reaction with the singlet oxygen generated, Fig. 6. For the complex, significant decrease in the DPBF absorption is observed at concentration ratios of DPBF to complex of ca. 20 to 1.
39 0.4
0.3
0.2 Absorbance
0.1
0.0 300 400 500 600 700 800 , nm
Figure 19: Time-dependent generation of singlet oxygen upon irradiation at l > 550 nm. [DPBF] = 20 mM, [I] = 0.85 mM.
Conclusion
A new synthetic process has been employed to expand on previous solvent-free synthesis of novel dipyrromethenes. In this process we have demonstrated that by adding TFA, previously elusive dipyrromethenes can be obtained in decent yield. Complex I showed unique spectroscopic properties with high molar absorptivities at wavelengths beyond 600 nm. Complex I also demonstrated the ability to generate singlet oxygen when irradiate at energies within the PDT window.
Chapter 3 References
1. Loudet, A.; Burgess, K. Bodipy dyes and their derivatives: syntheses and spectroscopic properties. Chem. Rev. 2007, 107, 4891-4932.
2. Lu, H.; Mack, J.; Nyokong, T.; Kobayashi, N.; Shen, Z. Optically active Bodipy’s. Coord.
Chem. Rev. 2016, 318, 1-15.
40 3. Odobel, F.; Zabri, H. Preparations and characterizations of bichromophoric systems composed of a ruthenium polypyridine complex connected to a difluoroborazaindacene or a zinc phthalocyanine chromophore. Inorg. Chem. 2005, 44, 5600-5611.
4. Galletta, M.; Campagna, S.; Quesada, M.; Ulrich, G. Ziessel, R. The elusive phosphorescence of pyrromethene-BF2 dyes revealed in a new multicomponent species containing Ru(II))- terpyridine subunits. Chem. Comm. 2005, 4222-4224.
5. Pandey, R.K. Recent advances in photodynamic therapy. J. Porph. and Phthalocyan. 2000, 4,
368-373.
6. Georgakoudi, I. and Foster, T.H. Singlet oxygen-versus non-singlet oxygen mediated mechanisms of sensitizer photobleaching and their effects on photodynamic dosimetry.
Photochem. Photobiol. 1998, 67, 612-625.
41 PART 2: THE IMPACT OF THE 515 NM EFFECT IN PHOTOSYNTHESIS: MODEL
SYSTEM USING β-CAROTENE ACID COMPLEXES
42 ABSTRACT
THE IMPACT OF THE 515 NM EFFECT IN PHOTOSYNTHESIS: MODEL
SYSTEM USING β-CAROTENE ACID COMPLEXES
Name: Wertz, Ashlee Elizabeth University of Dayton
Advisor: Dr. Mark B. Masthay
β-carotene (βC) is an orange pigment present in the photosynthetic reaction center (PRC) of green
1 plants, where it plays a vital role in photosynthesis: It quenches singlet oxygen ( O2, a toxic
1 oxidizing species generated during photosynthesis) before the O2 damages chlorophyll and other components of the PRCs. During photosynthesis, βC temporarily converts from its native orange–450 state to a pink–515 state via the so–called 515nm Effect. Because of the differences between the electronic structures of orange–450 and pink–515, my working hypothesis was that
1 pink–515 will quench O2 less efficiently than orange–450. This hypothesis has not been tested to date because orange–450 and pink–515 states are both present during photosynthesis, making
1 deconvolution of their relative O2 –quenching efficiencies effectively impossible. The objective of this research was to characterize the relative efficiencies with which “native orange” βC (λmax max = 450 nm), “acid–blue” βC complexes (λmax max 700 nm), and the transient “515nm–pink”
1 (λmax max = 515 nm) βC species generated during photosynthesis quench O2 and harvest blue and green light, with a view to understanding the impact of the 515 nm Effect on photoprotective and
1 light–harvesting roles of βC in photosynthesis. Through studies with the O2 substrate DBPF it
1 was found that the “acid-blue” βC was less efficient in quenching O2 than the “native-orange”
βC, but not to the extent that we expected. These experiments were not conclusive as there was a
1 large standard deviation in each set of data. We plan to repeat these experiments with a O2 detector (Ocean Optics NIRQuest) to obtain clean and more conclusive data. To date, we have
43 1 been able to generate a O2 signal at 1,270 nm using the device but have not yet fully optimized the experimental parameters.
44 ACKNOWLEDGMENTS
Many thanks to my research advisor Dr. Mark Masthay for guiding me through this project.
Thank you for the years of support and encouragement and for always having a positive attitude.
Additionally, I would like to thank my lab partners Lauren Hoody and Jackson Huang for their help with this work and Dr. Perry Yaney for his assistance with the optical devices. Finally, thank you to The University of Dayton Chemistry Department for funding this project and supporting me financially while I conducted research.
45 TABLE OF CONTENTS: PART 2
ABSTRACT………………………………………………………………………………………43
ACKNOWLEDGMENTS………………………………………………………………………..45
LIST OF FIGURES ……………………………………………………………………………...47
LIST OF TABLES ……………………………………………………………………………….48
CHAPTER 1 INTRODUCTION TO β-CAROTENE AND THE 515 NM EFFECT …………...49
Chapter 1 References ……………………………………………………….…………...51
CHAPTER 2 EXPERIMENTAL ………………………………………………………….……..53
Experimental Design …………….…………………………………………….………...53
Purification of βC using Gravity Filtration …………………….…………….…...... 53
CHAPTER 3 DPBF STUDIES ……………………………………………………...…………...55
CHAPTER 4 NIR-QUEST STUDIES ………………………………………...………………....58
Future Studies ………………………………………………….………...….…………..59
APPENDIX ……………………………………………………………………….……………...60
Choice of Acid ……………………………………………………………….….………60
Choice of Solvent ……………………………………………………………...... ………62
Vacuum Studies ………………………………………………………………….……...62
Appendix References ……………………………………………………………………64
46 LIST OF FIGURES
Figure 1. Structure of β- carotene ………………………………………………………………..49
Figure 2. Absorption spectrum of 10-5M βC in benzene solvent ……………………...... 51
Figure 3: Resulting slopes of DPBF experiments ………………………………………………..57
1 -3 Figure 4. Spectra from NIRQuest showing O2 signal at 1,270 nm with 10 M C60 .……………59
Figure 5. Decrease in visible absorption band of DPBF in the presence of TFA ………………..61
Figure 6. 10 -5 βC in benzene …………………………………………………………...... 63
Figure 7. 10 -5 βC and TFA after vacuum had been pulled ………………………….…………...63
47 LIST OF TABLES
Table 1. Solutions prepared and experimental first order rate constants obtained ………………56
48 CHAPTER 1
INTRODUCTION TO βC AND THE 515 NM EFFECT
β- Carotene
Carotenoids play two essential roles in photosynthesis: They (1) protect plants from
1 oxidative damage by quenching the toxic singlet oxygen ( O2) species generated when chlorophyll absorbs light; and (2) increase the effective wavelength range of photosynthesis by harvesting light energy in the blue-green (450-625 nm) region of the spectrum, where chlorophyll does not absorb 1. Carotenoids absorb in the blue-green portion of the visible spectrum; they are consequently yellow, orange or red in appearance and are responsible for the orange color of many “yellow” fruits and vegetables 2. In addition, “red–shifted” carotenoids are responsible for the brilliant green and blue coloration of some birds and crustaceans. Carotenoids such as the prototypical carotenoid β–carotene (Figure 1) serve as metabolic precursors of vitamin A and play an important role in human vision 3.
Figure 1: Structure of β- carotene.
515 nm Effect
β–carotene is also present in the photosynthetic reaction centers (PRCs) of green plants
1 where it plays an essential photoprotective role: βC rapidly “quenches” O2, thereby preventing oxidative damage to the PRCs. In plants, algae and photosynthetic bacteria, the primary electron donors and electron acceptors of the photosynthetic complexes are located on opposite sides of
49 the membrane. Light exposure results therefore in a charge separation across the photosynthetic thylakoid membrane due to photochemistry of the reaction centers and electron flow in the cytochrome b6f. The consequent movement of electrons and protons through the membrane generates a proton motive force (pmf) which gives rise to an electric field and a proton concentration gradient. The presence of the electric field influences the photosynthetic pigments the spectrum of which is modified owing to the Stark effect. The main consequence of chromophores exposed to an electric field is a shift of their absorption maxima 4. In its native orange–450 state, βC has a wavelength absorption maximum (λmax) of 450 nm. The λmax increases by 65nm as βC converts from the orange–450 state to a transient pink–515 state (λmax = 515nm) for approximately 0.01 seconds during the photosynthetic cycle when exposed to this electric
5-7 1 field . This so–called 515nm Effect is expected to decrease the O2–quenching efficiency ΦQ of
βC during the brief periods in the photosynthetic cycle when βC is in its pink–515 state;
푝푖푛푘−515 표푟푎푛푔푒−450 consequently Φ푄 < Φ푄 .
Since the 515nm Effect is inherent to photosynthesis, both the native orange–450 and
1 pink–515 states of βC contribute to O2–quenching in PRCs; in studies of green plants, then, it is
푝푖푛푘−515 표푟푎푛푔푒−450 not possible to distinguish Φ푄 from Φ푄 . Even so, by using model systems which—like the pink–515 state—are red–shifted with respect to the native orange–450 state, the
1 impact of the 515nm Effect on O2–quenching can be inferred. In this research, the pink–515 state was modeled using blue βC–Acid Complexes (βC–ACs)—in which the native orange–450 state of βC is converted to a blue–900 state by trichloroacetic as well as other acids 6.
50
Figure 2: (Black) Absorption spectrum of 10-5M βC in benzene solvent in its native orange-450 state. (Red) The same solution in the presence of 0.5M TFA, illustrating the acid-induced +450 nm bathochromic shift as βC converts to the blue-900 species. Figure courtesy of Lauren Hoody 2016 βC–ACs are particularly good models for the pink–515 state, for two reasons: (1) the red shift for
1 the blue–900 state is greater than that for the pink–515 state; and (2) the efficiency of O2– quenching is expected to vary inversely with the λmax of the quencher. Hence, the blue–900 state
1 was expected to be a poorer O2–quencher than the pink–515 state; making the difference in quenching efficiencies particularly easy to observe. The quenching efficiencies were thus
표푟푎푛푔푒−450 푝푖푛푘−515 푏푙푢푒−900 expected to follow the trend Φ푄 > Φ푄 > Φ푄 .
Chapter 1 References
1. Frank, H.A. and G.W. Brudvig, Functions of carotenoids in photosynthesis. Biochem., 2004. 43: p. 8607-8615.
2. Weedon, B.C.L., Occurrence, in Carotenoids, O. Isler, Editor. 1971, Birkhäuser: Basel. p. 29-59.
51
3. Liaaen-Jensen, S., Isolation, Reactions, in Carotenoids, O. Isler, Editor. 1971, Birkhäuser: Basel. p. 61-188.
4. Bailleul, B., Electrochromism: a useful probe to study algal photosynthesis. 2010, Photosynth
Res. p. 179-189.
5. Govindjee, Carotenoids in Photosynthesis: A Historical Perspective, in The Photochemistry of
Carotenoids, H.A. Frank, et al., Editors. 1999, Kluwer Academic Publishers: Dordrecht. p. 1-19.
6. Carr, F.H. and E.A. Price, Colour reactions attributed to vitamin A. Biochem. J., 1926. 20: 497-
501.
7. Haugan, J.A. and S. Liaaen-Jensen, Blue carotenoids. Part I. Novel oxonium ions derived from fucoxanthin. Acta Chem. Scand., 1994. 48: 68-75.
52
CHAPTER 2
EXPERIMENTAL
Experimental Design
-5 1 - Our solutions were made up of 10 M C60 (the O2 sensitizer) in benzene solvent with 10
5M βC in either the absence or presence of 0.5M TCA (a 50,000-fold TCA/ βC molar excess).
The line from a 532 nm pulsed Nd:YAG laser was used to irradiate these solutions to generate
1 1 O2. DPBF studies or a time resolved O2 detector were used to determine the amount of singlet oxygen generated by the solution.
Purification of βC Using Gravity Filtration
To prevent condensation of water vapor onto our samples, βC (Sigma-Aldrich Type I, synthetic, ≥ 93%) was moved from the freezer to a dark drawer and allowed to thaw to room temperature for 1 hour prior to use. 125 mL of solid silica gel was placed into a 250 mL beaker. A slurry of silica gel was created using benzene (Sigma-Aldrich, for HPLC ≥ 99.9%) and the mixture was packed into the column to a height of about 25 cm. ~500 mg of the unpurified βC(s) was dissolved in ~25 mL benzene. The resulting (nearly saturated) solution was then added a few drops at a time to the head of the column. Additional benzene was then added to the solvent head to force the concentrated βC solution into the top of the silica gel column more quickly. This process was repeated until all the dissolved βC had been forced onto the column, at which point a benzene head was maintained above the silica gel to facilitate elution through the column via gravity.
The initial clear-to-yellowish band was collected as waste until the prominent dark orange band of the βC began to leave the column. At this point, purified βC was collected in a different container. To optimize the purification, the purified βC eluent was only harvested for a
53
40 minute period. The purified solution was then stirred under a stream of compressed air or nitrogen at ~5 psi to evaporate the benzene. Evaporation was continued until the purified βC appeared dry. The purified βC was then warmed to room temperature to ensure that no benzene had frozen because of the evaporation. When benzene had frozen, the mixture was warmed to room temperature and this evaporation process was repeated until all the benzene was removed.
The final βC(s) product was stored in a septum–capped vial under an ~2 psi atmosphere of compressed argon (99.9998%) to prevent reaction with ambient O2 and stored in a freezer at 0⁰ C.
54
CHAPTER 3
DPBF STUDIES
1 2,6-Diphenylisobenzofuran (DPBF; Sigma-Aldrich, 97%) is commonly used as a O2
1 substrate. We accordingly attempted to use DPBF to quantify O2 in our solution to supplement our anticipated NIRQuest I1270 results detailed in chapter 4 below. However, we found that
1 quantification of O2 with DPBF may yield results of limited reliability. When yellow-blue solutions of DPBF in benzene were placed in quartz cuvettes, the solution turned colorless over time. In fact, upon exposure to 3 minutes of direct sunlight (mid-morning, July, Dayton, Ohio,
39.7589°N, 84.1916°W), the DPBF absorption peak was completely eliminated and the solution became colorless.
Similar results were obtained with ambient (overhead fluorescent) light (although the loss of absorbance and color was slower with ambient light). Surprisingly, the rate of degradation was
1 roughly the same in both aerated and sparged (i.e., deoxygenated) solutions, indicating that O2 does not play a role in this photodegradation process. The reaction appears to be a direct photodegradation reaction of DPBF, in which DPBF either (1) directly decomposes in an oxygen−and solvent−independent fashion; or (2) reacts with benzene. To confirm that the degradation is initiated by light, two cuvettes of DPBF in benzene were prepared; one was placed in a dark drawer and the other was placed in ambient light overnight. The next morning, the cuvette in the dark retained the same color as when it was originally prepared, whereas the cuvette that had been in the light had turned colorless. Clearly then, DPBF is photolabile in benzene.
The experimental results obtained also show the inconsistencies with using DPBF instead
1 of direct detection of O2. Solutions were prepared as detailed in Table 1. Each sample was irradiated with a 532 nm laser line from a Nd:YAG laser and absorption spectra were taken at a
55 series of time intervals. The first spectra were taken before irradiation and the time intervals at which the spectra were taken was consistent across all samples. The peak absorption from each spectrum for each solution was plotted and fitted to first order kinetics. The rate of degradation for each solution was determined and is represented as the slope in Table 1.
Table 1: Solutions prepared and experimental first order rate constants obtained.
Solution DPBF + C60 βC + DPBF + βC-TCA + DPBF + TCA + DPBF +
C60 C60 C60
Slope (sec -1) -0.0152 -0.00565 -0.0066 -0.01735
Standard Dev. 0.00646 0.00134 0.00172 0.00774
If my hypothesis is confirmed then the blue sample whose wavelength had been shifted by the acid (βC-TCA + DPBF + C60) shows a more negative slope than the orange sample that is in its native state (βC + DPBF + C60). This indicates that the species with the longer λmax is less
1 efficient at quenching the O2. This expectation is shown by the data, but not to the extent that was expected.
56
Figure 3: Resulting slopes of DPBF experiments after fitting decrease in DPBF absorption to first order rate kinetics.
An additional concern with the data in Table 1 is that there was a large amount of variation between different trials. Even under chemically-optimized conditions with care taken to repeat the exact procedure the data between trials was widely variant. This led to the conclusion that DPBF was influencing the system in ways other than acting as a single oxygen substrate. To get more definitive data, tests must be performed without DPBF and with use of a time resolved
1 O2 photodetector.
57
CHAPTER 4
NIR-QUEST STUDIES
표푟푎푛푔푒−450 푏푙푢푒−900 We plan to characterize Φ푄 and Φ푄 using a state-of-the-art transient near infrared emission spectrophotometer, more specifically, an Ocean Optics NIRQuest: a germanium photodiode array-based transient infrared spectrometer with an operating range of
900-1700nm. This detector was selected for four reasons. First, its operating range encompasses
1 the anticipated 1270nm O2 emission signal. Second, the NIRQuest interfaces easily with our pulsed Nd:YAG laser. Third, its cost ($16,000) was fundable using Chemistry Department funds.
Fourth, it is able to be cooled to temperatures low enough to minimize unwanted background blackbody radiation.
1 In these studies, the O2 generator buckminsterfullerene (C60; Sigma-Aldrich, 99.5%; used as purchased) was added to solutions of βC and serially diluted to concentrations of 10-5M
1 C60. O2 was generated by irradiating these βC + C60 and βC-TFA + C60 solutions with the 532nm second harmonic of a pulsed Nd:YAG laser (Spectra Physics INDI-40). The intensity I1270 of the
1 resulting 1,270nm emission of O2 emanating from these solutions was monitored using an Ocean
Optics NIRQuest Spectrophotometer operating in Edge Trigger mode (with 523 nm laser pulses).
We are currently in the process of optimizing this procedure. To date, we have successfully
1 -3 observed a clear O2 signal from 10 M C60.
58
1 -3 Figure 4: Spectra from NIRQuest showing O2 signal at 1,270 nm with 10 M C60.
To obtain this signal a lens had to be placed in front of the sample to narrow the beam to a horizontal line. In addition, certain parameters had to be put in place on the NIRQuest including having 40 scans to average, an integration time of 3 seconds, cooling the device with thermoelectric cooling to at least -20⸰ C and storing a dark spectrum before collecting data. The
-5 experimental parameters will have to be further optimized to show a signal with 10 M C60.
Future Studies
In the future we would like to fully optimize the NIRQuest parameters in order to get a
1 -5 clear O2 signal using 10 M C60 and perform the experiment outlined above in order to get conclusive results. In addition, it would be beneficial to perform Gaussian Molecular orbital
1 studies to see if these calculations are consistent with the proposed electronic structures of O2 and βC.
59
APPENDIX
Choice of Acid
The acid that was initially decided upon for this set of experiments was TFA (trifluoroacetic acid). This acid was chosen for a variety of reasons, mostly through process of elimination. The first acid that was tested was H2SO4 (Fischer scientific, Certified ACS Plus). However, issues arose with the separation that took place when this acid was placed into CH2Cl2. 0.005 g β- carotene was massed and diluted to 20 mL with DCM. With the addition of approximately 1 mL
H2SO4 the solution turned blue and immediately separated into a dark blue bottom layer and a lighter blue top layer. After 4 days the top layer was much clearer. The question was brought up as to which compounds were present in the bottom layer. The density of CH2Cl2 is 1.325g/cm^3 and that of H2SO4 is 1.84 g/cm^3 so the assumption was that H2SO4 was the compound that made up the bottom layer. To determine if CH2Cl2 was also present in the bottom layer 2 mL CH2Cl2 and and 8 mL H2SO4 were poured into a volumetric flask and left to sit for 2 days. There was a clear meniscus at the 2 mL mark and when the flask was returned to 2 days later the meniscus was still present at the same level. This helped prove that the CH2Cl2 and H2SO4 were not mixing and it was most likely only H2SO4 and β-carotene present in the bottom layer and no CH2Cl2. To address the color loss of the top layer a new 0.00025 mg/L solution was made and tested at different time intervals using a UV-VIS spectrometer. The spectrum shows a clear decomposition of the top layer over a time period of three hours. Because of the uncertainty that came with the
H2SO4 this acid was ruled out.
Next, 0.0025 g β -carotene was mixed with 10 mL CH2Cl2 and methanesulfonic acid
(CH3SO3H, Sigma-Aldrich, 99%) was added. The result was a dark blue color that separated into a darker layer on top and a light, brownish layer on bottom. This acid was ruled out because of that same issue with separation that was seen with H2SO4. Nitric acid (HNO3; Fischer scientific,
60
Certified ACS Plus) was tested through the same process and the result was a bright blue color, however, this blue compound turned to an orange-brown top layer and a clear bottom layer over time consistent with reports in the literature 1. Trifluoroacetic acid was added to 0.0025 g β - carotene in 10 mL CH2Cl2 in the same manner as the other acids and the result was a dark blue color. TFA did not cause the mixture to separate into two separate layers so it was decided that this would be the acid used in the remainder of the experiments. However, it was later discovered that DPBF degrades in the presence of TFA. This result is consistent with reports in the literature, which indicate, that in ambient light, TFA causes the intensity of the visible absorption band of
DPBF to lose its absorption peak at a rate that is proportional to TFA 2.
Figure 5: Decrease in visible absorption band of DPBF in the presence of TFA
This decrease in absorption peak was not seen with trichloroacetic acid (TCA), therefore, it was determined that both the DPBF and NIRQuest studies would be done with the use of TCA.
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Choice of solvent
Initially, the majority of the solutions were prepared in CH2Cl2 (CHROMASOLV® Plus, for
HPLC, ≥ 99.9%) solvent because βC and C60 are both soluble in CH2Cl2. Unfortunately, however,
3-5 βC photodegrades in CH2Cl2 . This was seen with both overhead, room lights and outdoor, sunlight. Hence, benzene (Sigma-Aldrich, for HPLC ≥ 99.9%) was our ultimate solvent choice for two reasons: (1) βC and C60 are both soluble in benzene; and (2) βC is photostable in benzene.
Vacuum Studies
Vacuum studies were done to determine the nature of the interaction between the βC and TFA.
0.05 g βC was massed and added to 10 mL benzene. 1 mL of this sample was taken and added to
100 mL additional benzene making a 10 -5 M beta carotene solution. This solution was then poured into 5.7 g TFA making a 50,000 M excess TFA solution. With the addition of TFA the orange solution turned a dark blue, as was expected. The mixture was poured into a round bottom flask and solvent was vacuumed out. When vacuumed the solution froze and the flask was warmed by hand to speed the process. When all of the solvent had been evaporated out there was an orange oily ring along the flask with a red powder settled at the bottom. The color change is important to note. The loss of the characteristic blue color could provide evidence for the TFA being pulled out by the vacuum which could mean the reaction between the TFA and βC is a weak one. The residue that was left behind was dissolved in additional benzene and a UV-VIS spectrum was taken of this sample. Even though both βC in benzene and the result of this experiment result in the same characteristic orange color the spectra are not identical. Both compounds show absorbance in the same wavelength region, however, the peaks are not equivalent.
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Figure 6: 10 -5 βC in benzene
Figure 7: 10 -5 βC and TFA after vacuum had been pulled
A TLC plate was spotted to test for impurities in the compound that had been obtained by vacuum. This plate showed that there were indeed some impurities present. The nonpolar compounds moved along the plate with the solvent front and the polar compounds were left at the
63 bottom leaving two distinct spots. Using preparative thin layer chromatography, the nonpolar compounds were scraped off the TLC plate and dissolved in benzene. This spectrum once again showed an absorbance that was around the same wavelength as βC in benzene but with different peaks. However, this does not necessarily mean that there was no pure βC present in the sample obtained by the vacuum. Because the sample moved all the way up the solvent front it is not necessarily guaranteed that the nonpolar sample is one, pure compound. There could be more than one nonpolar substance present, one of which could be pure βC.
Appendix References
1. Karrer, P. and E. Jucker, Carotenoids, E.A. Braude, Editor. 1950, Elsevier Publ. Co.: New
York.
2. Zhang, X.F. and X. Li, The photostability and fluorescence properties of diphenylisobenzofuran. J. Luminescence, 2011. 131: 2263-2266.
3. Wang, W., Photochemical Characterization of the Intensity Dependence in Multiphoton-
Reactive Systems: Application to the Photodegradation of β-Carotene, 2008M.S. Thesis,
Chemistry, University of Dayton, Dayton, OH.
4. Gurzadyan, G.G. and S. Steenken, Photoionization of β-carotene via electron transfer from excited states to chlorinated hydrocarbon solvents. A picosecond transient absorption study. Phys.
Chem. Chem. Phys., 2002. 4(13): 2983-2988.
5. Fujii, R., Y. Koyama, A. Mortensen and L.H. Skibsted, Generation of radical cation of β- carotene in chloroform via the triplet state as revealed by time-resolved absorption spectroscopy.
Chem. Phys. Lett., 2000. 32
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