Small Molecule Probes for Photodynamic Therapy (PDT)

by

Kamalpreet Singh

A thesis submitted in conformity with the requirements for the degree of Master of Science

Department of Chemistry University of Toronto

© Copyright by Kamalpreet Singh 2019

ii

Small molecule probes for photodynamic therapy (PDT)

Kamalpreet Singh

Master of Science

Department of Chemistry

University of Toronto

2019

Abstract

Photodynamic therapy is a clinically approved cancer treatment that utilizes singlet oxygen

generated via a combination of photosensitizer (PS), light and molecular oxygen to kill cells.

Unfortunately, the technique remains under-utilized due to the indiscriminating phototoxicity of

PSs upon irradiation, causing damage to healthy cells alongside cancer cells. Pro-drug derivatives

of pre-existing PSs that are activated by enzymes overexpressed in cancer cells offer a great

solution. Such derivatives are inactive until acted upon by cancer biomarkers, allowing for cancer

targeted cell death. This work highlights the preliminary work in the development of 2 such

activatable PSs, activated by the cancer biomarkers, O6-methylguanine-DNA-methlytransferase

and Nitroreductase. In addition, the development and photophysical characterization of a novel

coumarin based PS is also discussed.

iii

Acknowledgments

I would like to begin by thanking my thesis supervisor and mentor, Dr. Andrew Beharry. Thank

you for giving me the opportunity to work in your lab and helping me grow as a scientist. I hope

to continue using the skills you have given me to make you proud wherever I go.

I would also like to thank Dr. Patrick Gunning for serving as the second reader for this thesis. I am

grateful for your valuable feedback and mentorship in my time of need.

I also want to add a special mention to my family and friends (Angel, Khalid, Aditya, Advait,

Dave, Fioralba, Aaron, Johan and many more not listed here) for their continuous emotional and

moral support. The companionship of these individuals in my first year did not let me feel the

absence of a lab group.

Finally, I would like to thank the people at the Beharry lab. There was never a dull moment

working in the lab, making it feel like home. I would like to especially thank Rita Bodagh for

working on the MGMT protein expression with me and teaching me mammalian culturing. I would

also like to thank Karishma Kailass for conducting the cell imaging for the MGMT project and

Nima Gharibi for measuring the irradiation intensity.

iv

Table of Contents

Acknowledgments ........................................................................................................................ iii

Table of Contents ......................................................................................................................... iv

List of Figures ............................................................................................................................... vi

Chapter 1 Photodynamic Therapy ...............................................................................................1

Cancer .........................................................................................................................................1

What is photodynamic therapy (PDT)?.......................................................................................2

Photosensitizers (PSs) .................................................................................................................4

Activatable Photosensitizers (PSs) ............................................................................................11

Mechanisms of quenching & activatable photosensitizers .......................................................12

Chapter 2 On the road to developing an MGMT activatable Photosensitizer .......................18

O6-Methylguanine DNA Methyltransferase (MGMT) as a PDT target....................................18

Results and discussion ..............................................................................................................22

Future directions & experiments ...............................................................................................31

Materials & methods .................................................................................................................33

Chapter 3 A Hypoxia activatable Photosensitizer ....................................................................44

Nitroreductase (NTR) as a PDT target ......................................................................................44

Results and discussion ..............................................................................................................46

Future directions & experiments ...............................................................................................53

Materials & methods .................................................................................................................54

Chapter 4 3AIC: A Novel Coumarin Based Photosensitizer ...................................................57

Why does the world need a coumarin photosensitizer (PS)? ....................................................57

Results and discussion ..............................................................................................................58

Future directions & experiments ...............................................................................................64

v

Materials & methods .................................................................................................................65

Concluding Remarks ...................................................................................................................69

References .....................................................................................................................................72

Appendix A: NMR & Mass Spectrum of Synthesized Compounds ........................................78

vi

List of Figures

Figure 1.1. A simplified Jablonski diagram for PSs ....................................................................... 2

Figure 1.2. Type I and Type II excitation pathways in PSs ............................................................ 4

Figure 1.3. The biological optical window ..................................................................................... 5

Figure 1.4. Structures of Porphyrin, Chlorin, Bacteriochlorin, and Phthalocyanine ...................... 6

Figure 1.5. Structures of Phenothiazine, xanthene and bodipy scaffolds. ...................................... 7

Figure 1.6. A schematic of the mechanism of action of an activatable PS. .................................. 12

Figure 1.7. A schematic of the mechanism of FRET. ................................................................... 13

Figure 1.8. A schematic of the mechanism of activation of a FRET based activatable PS ......... 14

Figure 1.9. An overview of the quenching due to photoinduced electron transfer (PeT).. .......... 15

Figure 1.10. A schematic of the mechanism of activation of a PeT based activatable PS ........... 16

Figure 1.11. A schematic of the mechanism of activation of a ICT based activatable PS .......... 16

Figure 1.12. A schematic of the mechanism of activation of a caged activatable PS ................. 17

Figure 2.1. A crystal structure of Human MGMT (E.C. 2.1.1.63) .............................................. 18

Figure 2.2. Mechanism of DNA repair by MGMT ....................................................................... 19

Figure 2.3. Expression levels of MGMT in cancer and healthy tissues ....................................... 20

Figure 2.4. Inhibition of MGMT by O6BG & Patrin-2 ................................................................ 20

Figure 2.4. Inhibition of MGMT by O6BG & Patrin-2 ................................................................ 20

Figure 2.5. Modifications of O6BG tolerated by MGMT ............................................................ 20

Figure 2.6. Mechanism of activation of a FRET based MGMT activatable PS .......................... 21

Figure 2.7. Structure of P1 (probe 1) ........................................................................................... 22

vii

Figure 2.8. The synthetic scheme of probe 1 (P1) ........................................................................ 24

Figure 2.9. UV-Visble spectrum of P1 ......................................................................................... 23

Figure 2.10. Fluorescence excitation spectra of P1 ...................................................................... 25

Figure 2.11 Fluorescence emission spectra of P1 ......................................................................... 25

Figure 2.12. A schematic of the mechanism of activation of P1 by MGMT. ............................... 26

Figure 2.13. Fluorescence activatability assay ............................................................................. 26

Figure 2.15. The structure of probe 1 no quencher (P1NQ). ........................................................ 27

Figure 2.16. The synthetic scheme for P1NQ. .............................................................................. 28

Figure 2.17. Initial (test) SDS PAGE assay .................................................................................. 29

Figure 2.18. SDS PAGE assay. .................................................................................................... 30

Figure 2.19. Images from wash out in cellulo assay ..................................................................... 31

Figure 2.20. Future directions ...................................................................................................... 32

Figure 2.21. A proposed PeT based MGMT activatable PS probe (O6BG-PL-3AIC). ................ 33

Figure 3.1.Type I and Type II NTR .............................................................................................. 45

Figure 3.2. Mechanism of the NTR mediated activation of p-NBMB ......................................... 46

Figure 3.3. The synthetic scheme for p-NBMB ............................................................................ 47

Figure 3.4. p-NBMB activatability assay ..................................................................................... 47

Figure 3.5. A kinetic p-NBMB activatability assay (with NTR) .................................................. 48

Figure 3.6. A kinetic p-NBMB activatability assay (without NTR) ............................................. 48

Figure 3.7. Mechanism of activation for the singlet oxygen sensor green (SOS-G) .................... 50

viii

Figure 3.8. p-NBMB singlet oxygen assay (with NTR) ............................................................... 50

Figure 3.9. p-NBMB singlet oxygen assay (without NTR) .......................................................... 50

Figure 3.10. Mechanism of activation of the ROS sensor (DCFH-DA) ....................................... 52

Figure 3.11. p-NBMB ROS assay (without NTR) ....................................................................... 50

Figure 3.12. p-NBMB singlet oxygen assay (with NTR) ............................................................. 50

Figure 4.1. Structure of Eosin ....................................................................................................... 50

Figure 4.2. Structure of Psoralen .................................................................................................. 50

Figure 4.3. Synthetic scheme for the synthesis of 3AIC ............................................................... 59

Figure 4.4. The UV-Visible spectrum of 3AC and 3AIC ............................................................. 59

Figure 4.5 3AIC singlet oxygen assay ......................................................................................... 60

Figure 4.6. 3AC singlet oxygen assay .......................................................................................... 61

Figure 4.7. 3AIC ROS assay ........................................................................................................ 62

Figure 4.8. 3AC ROS assay .......................................................................................................... 63

Figure 4.9. 3AIC Photostability Assay ........................................................................................ 64

Figure 5.1. Structure of the examined enzyme activatable PS probes. ......................................... 69

Figure 5.2. The structure of probe 1 no quencher (P1NQ). .......................................................... 70

Figure 5.3. Structure of the 3AIC. ................................................................................................ 71

Figure S1. 1H-NMR of guanine salt in DMSO-d6. ....................................................................... 78

Figure S2. 1H-NMR of Alkyne O6 Benzyl Guanine in DMSO-d6. .............................................. 79

Figure S3. 1H-NMR of Alkyne O6 Benzyl Guanine N9 Propylamine BHQ2 in DMSO-d6. ........ 80

ix

Figure S4. 1H-NMR of 3-azidopropan-1-ol in CDCl3. ................................................................. 81

Figure S5. 1H-NMR of 3-azido-N-methylpropan-1-amine in CDCl3. Note, the oil was

contaminated with THF and TEA. ................................................................................................ 82

Figure S6. 1H-NMR of CF3 protected 4-methylamine benzyl alcohol in CDCl3. ......................... 83

Figure S7. 1H-NMR of CF3 protected 4-methylamine O6 benzyl guanine in DMSO-d6. ............ 84

Figure S8. 1H-NMR of N-acetylglycine in DMSO-d6. ................................................................ 85

Figure S9. 1H-NMR of 3AIC-Acetate in DMSO-d6. ................................................................... 86

Figure S10. 1H-NMR of 3AIC in CDCl3. ..................................................................................... 87

Figure S12. ESI- mass spectrum of probe 1 (P1) dissolved in methanol. ..................................... 89

Figure S13. ESI+/ESI- mass spectrum of Sarcosine linked pyro A dissolved in methanol. ......... 90

Figure S14. ESI+ mass spectrum of probe 1 no quencher (P1NQ) dissolved in methanol. .......... 91

Figure S15. ESI+ mass spectrum of p-NBMB dissolved in methanol. ......................................... 92

1

Chapter 1 Photodynamic Therapy

Cancer

As the second leading cause of deaths in the world, cancer has become a global health problem1,2.

In fact, the uncontrolled cell division characteristic of the family of more than 100 diseases called

cancer, is the primary cause of deaths in Canada; killing about 1 in 4 Canadians3,4. To treat this

ailment, 3 key traditional treatment regiments - surgery, radiotherapy and chemotherapy - are

commonly employed5,6.

In the surgical approach, the malignant tumor body is excised from the patient using surgical tools7.

Unfortunately due to the local nature of this approach, it is restricted to the treatment of solid

tumors or tumors that are constrained in one area7. Therefore, surgical methods cannot be

efficiently employed in the treatment of disseminated cancer or metastasis, that is the spread of the

tumor from the primary site to distal secondary sites5,7,8. In addition, the cuts produced in the body

from the extraction of the tumor can be painful and slow to heal post-surgery. Consequently,

individuals may opt to utilize an alternative local treatment approach called radiotherapy. Like the

surgical method, radiotherapy also targets localized tumor bodies9. However, instead of utilizing

scalpels, radiotherapy exploits the DNA damaging property of high energy radiation to induce cell

death9. This type of treatment is often utilized in combination with other approaches

(chemotherapy and surgery) to minimize chances of cancer returning9. Unfortunately, much like

surgery, the local nature of radiotherapy also prevents its’ utilization in the issue of metastasis10.

In addition, the exposure of the healthy tissue surrounding the malignant tumor to high energy

radiation during the therapy, often results in severe side effects9. Due to the local restrictions and

adverse effects associated with surgery and radiotherapy, a third type of cancer treatment called

chemotherapy is often called upon5,8. Chemotherapy (“chemo”) is a cancer treatment approach

built around the cytotoxic nature of chemical agents5,8. Due to the lack of local restrictions,

chemotherapy is often utilized for the treatment of disseminated or metastasized cancer5,8.

Although chemo can get around the issue of localized action, anti-cancer drugs frequently lack

structural features which allow them to distinguish between cancer and healthy tissue, leading to

adverse effects in healthy cells alongside tumor cell death11,12. In addition, the success of many

chemo agents is impeded by a loss in potency due to built-in mechanisms in the cells, i.e.

2

resistance8,10. Evidently, the 3 traditional approaches have major limitations and drawbacks and so

it is not surprising that the prognosis for cancer patients remains suboptimal.

What is photodynamic therapy (PDT)?

PDT is a minimally invasive clinically approved cancer treatment approach that relies on the

combined action of 3 individually innocent components - a photosensitizer (PS), light, and

molecular oxygen - to induce cell death13–19. In comparison to traditional treatment regiments, PDT

has several advantages including its non-invasive nature, little to no scarring, ability to treat the

patient in an outpatient setting, ability to utilize repeated doses without the issue of resistance or

exceeding total dose limitations, and fewer adverse effects in comparison to chemotherapy13,14,19.

The process begins with administration of the PS which may be topical or systemic14. Upon

accumulation of the PS in tissues, the target tissue is irradiated with an appropriate wavelength of

light, which results in the excitation of the PS to a singlet excited state. In the singlet excited state,

the excess energy carried by the PS can be dissipated via several processes (Figure 1.1). The excess

energy can be lost via emission of light (fluorescence) or by heat (internal conversion).

Alternatively, the excess energy may also be lost via interactions of the PS dye with its’

surroundings (quenching). The PS can also undergo an electronic rearrangement changing its spin

from the singlet excited state to the triplet excited state. In the triplet-excited state the PS can also

lose the excess energy via emission of light (phosphorescence), heat (internal conversion), or by

various quenching mechanisms.

Figure 1.1. A simplified Jablonski diagram describing the photophysical processes that occur after excitation

of a PS.

3

The excited triplet-state PS can also interact with molecules in its’ surroundings such as molecular

oxygen via non-radiative energy transfer processes, converting ground state triplet oxygen to an

excited singlet state (Figure 1.1)13,17. This type of energy transfer between a PS and molecular

oxygen is referred to as a Type II process (Figure 1.2)13,17. If in a cellular environment, the resultant

reactive form of oxygen can oxidatively damage cells causing cell death. Due to the short half-life

(10-320 nanoseconds) and diffusion (10 to 55 nm) of singlet oxygen in cells, the damage produced

by PSs is often limited to the site of localization, which in combination with intensity of irradiation

and concentration of PS (PDT dose), plays an active role in the mode of cell death13–16,18.

Traditionally it was believed that PDT induced cell death via either apoptosis or necrosis13. PSs

which accumulated in the mitochondria and endoplasmic reticulum (ER) were attributed to cell

death via apoptosis, whereas those which accumulated in the lysosomes or plasma membrane to

necrosis13. However, recent studies have shown that the PDT dose plays a crucial role in the form

of cell death. Photooxidative damage induced by high PDT-dosage in organelles and plasma

membrane are thought to induce ATP depletion thereby initiating necrosis14–16. Whereas, cell

death brought on by low PDT-dosage is thought to be a result of autophagic cell death14–16. In

addition, reactive oxygen species (ROS) production in mitochondria, endoplasmic reticulum (ER),

Golgi apparatus, and even lysosomes are thought to initiate apoptotic cell death14–16.

In addition to the type II processes, PSs can also participate in a Type I process, whereby the PS

undergoes a radical reaction with organic molecules in the cell microenvironment (Figure

1.2)13,16,17. The electron/hydrogen transfer reactions between cellular components and the PS can

result in the generation of a complex mixture of ROS such as superoxide anion radical, hydrogen

peroxide, and hydroxyl radical13,15,16. These ROS species can oxidatively damage the cell, causing

death. Due to the several species involved, type I processes are generally more mechanistically

complex compared to type II processes16. Most PSs are believed to act via type II mechanisms,

with singlet oxygen being the primary cytotoxic agent13,16. Evidently, the properties of the PS play

an integral role in the type of active cytotoxic agent and the mode of cell death. In this regard, the

choice of the appropriate PS is vital in achieving optimal efficacy for treatment.

4

Figure 1.2. A schematic of the two pathways by which a photosensitizer (PS) excited by the approviate

wavelength of light (h), can induce oxidative damage in biomaterials (BM). This image was obtained from

[17].

Photosensitizers (PSs)

As highlighted above, the PS is perhaps the most significant component of PDT. In this context, a

PS refers to a chemical agent or drug, which functions as a mediator between light energy from an

illumination source and molecular oxygen in the cells. To be more specific, the PS is a

chromophore which can absorb light of a particular wavelength and thereafter transmit that excess

light energy on to an oxygen molecule, generating singlet oxygen which can kill cells. The ideal

PS should exhibit minimal dark toxicity, that is it should not be toxic until irradiated with

light14,17,20. The PS should also have good amphilicity17,21. In other words, the PS should have

enough hydrophilicity such that upon systemic administration, the PS can travel to the desired

location with minimal aggregation and degradation but, should also be lipophilic enough to

penetrate the cellular membrane. In addition, it is desirable to have a PS which can be activated by

light energy that falls within the biological optical window (650-900nm) where the absorbance

from endogenous chromophores such as hemoglobin and water is minimal (Figure 1.3)14,17,20,22,23.

Other desirable traits in a PS include high photoactivity (high singlet oxygen quantum yield), high

chemical and photostability and rapid clearance to minimize phototoxic side effects post

treatment14,17,20. The search for such an ideal PS has led to the development of more than 400

chromophores, however, no PS that satisfies all the desired traits has yet been found19.

5

Figure 1.3. The biological optical window (“in vivo imaging”) refers to the region of the visible spectrum

spanning 650-900nm where endogenous chromophores exhibit lowest absorbance. This Figure was obtained

from [22].

Based on their structures, PSs can be divided into two different classes; tetrapyrrole based PSs and

non-tetrapyrrole PSs. The tetrapyrrole PSs family is built around the heterocyclic aromatic ring

structure of 4 different supermolecules: porphyrins, chlorins, bacteriochlorins and an isoindole

derivative called Phthalocyanines (Figure 1.4). Porphyrins are highly attractive chromophores for

PDT, as they absorb in the red (610-630 nm) region of the visible spectrum and exhibit strong

fluorescence upon excitation at the Soret band (~ 400 nm)19. In fact, the porphyrin class was the

first set of compounds used for PDT in the form of a complex porphyrin oligomer mixture called

Photofrin (Table 1.1)20,24. Photofrin was clinically approved for PDT in 199318,20,24. Although still

widely in use, Photofrin suffers from several disadvantages such as prolonged skin sensitivity

(poor clearance) and a small absorbance peak at 630 nm (ε630 ~3000 M-1 cm-1)20,24. Consequently,

other porphyrin-based PSs have now been developed to improve upon these limitations. As shown

in Table 1.1, examples include pyropheophorbide-a, which has a much greater absorbance in the

red region (ε665 ~46000 M-1 cm-1) and protoporphyrin-IX (ε626 ~3000 M-1 cm-1) which exhibits less

skin sensitivity (metabolized in 48 hours)13,20,24.

6

Figure 1.4. Chemical structures of Porphyrin, Chlorin, Bacteriochlorin, and Phthalocyanine macromolecules.

The reduction of one double bond in a pyrrole ring of the porphyrin scaffold leads to another key

class of tetrapyrrole based PSs called Chlorins. These are popular scaffolds for PSs as they have a

high quantum yield of singlet oxygen and a stronger absorption in the red (650-660nm) region

relative to porphyrins coupled with a small bathochromic shift19. Examples of clinically approved

chlorin based PSs include Foscan (m-tetrahydroxyphenylchlorin), Verteporfin (benzoporphyrin)

and chlorin-e6 (Table 1.1)20,24. A further reduction of a double bond in the pyrrole ring of the

chlorin structure leads to another subset of tetrapyrrole PSs called bacteriochlorins

(tetrahydroporphyrin). Due to the double reduction of the pyrrole rings, bacteriochlorins have a

strong absorbance in the 700-800 nm range, a significant bathochromic shift relative to porphyrins

and chlorins19. Consequently, bacteriochlorins offer greater tissue penetration in comparison with

porphyrins and chlorins and as such are an attractive target for utilization in PDT. Examples of

clinically approved bacteriochlorin PSs include Bacteriochlorophyll-a (Table 1.1)13. Another

approach that has been utilized to improve the poor absorbance of porphyrins in the red region of

the visible spectra, is the development synthetic analogues employing isoindole heterocyclic rings

instead of pyrroles. The greater aromatic nature of these scaffolds called Phthalocyanines, resulted

in an incredible increase in the light absorption properties relative to porphyrins, with the scaffolds

having extinction coefficients as great as 200,000 M-1cm-1 in the 650-700 nm range19,20. An

example of a clinically approved Phthalocyanine is aluminum phthalocyanine tetrasulfonate

(Table 1.1)13,20,24. Although, the tetrapyrrole class of PSs exhibit great singlet oxygen quantum

yields and long-wavelength absorbance, many members of this family suffer from poor clearance

leading to prolonged skin sensitivity19,20. In addition, due to the bulky hydrophobic nature of

several members of this class of PSs, they are often prone to aggregation in aqueous environments

hindering systemic administration19,20. Therefore, numerous different non-tetrapyrrole PSs based

7

upon natural and synthetic dyes have been developed and investigated for their photodynamic

activity including hypericin, phenothaizines, xanthenes, and BODIPY.

Hypericin is a naturally occurring chemical that can be extracted from St. John’ wort and is well

known for its ability to make ROS upon excitation in the orange part of the visible spectrum19,20,24.

It has been shown to accumulate in greater amounts in tumor bodies relative to surrounding healthy

tissue and as such is an attractive PS for clinical PDT19. Unfortunately, the extremely hydrophobic

structure of hypericin prevents its utilization as a standalone small molecule PS, requiring a

delivery vehicle such as liposomes24. Clinical investigation of hypericin drug vehicle formulations

to date have been unsuccessful and further investigation is underway20.

Phenothaizines are a family of synthetic dyes derived from the

phenothiazine scaffold (Figure 1.5). Derivatives of this family have

exhibited various biological activities, and as such are actively

employed in the field of medicine and histology. Examples of PSs

derived from the phenothiazine family include methylene blue (MB)

and toluidine blue O (TBO) (Table 1.1)13,19,20,24. These water-soluble

dyes have high singlet oxygen quantum yields and are photoactivated

in the biological optical window (665 nm for MB and 630 nm for

TBO)13,19,20,24. Unfortunately, the positive aspects of MB and TBO

are impeded by their dark toxicity which induces adverse side

effects19. Nevertheless, the clinical potential of these PSs for cancer

PDT is being currently investigated20.

Another group of synthetic dyes that have found their way into the realm of PDT, are the

xanthenes. Much like phenothaizines, the core xanthene scaffold (Figure 1.5) has also found much

utility through derivatization, serving as the core structure in many different fluorophores such as

fluorescein and rhodamine. Further manipulation of the fluorescein structure via introduction of

various heavy atom halogens to promote intersystem crossing has allowed the development of

effective PSs such as Rose Bengal (Table 1.1)13,19,20,24. Rose Bengal is a water-soluble PS which

is photoactivated in the green (560nm) part of the visible spectrum13. It has both a high

fluorescence quantum yield and singlet oxygen quantum yield13. Unfortunately, Rose Bengal

exhibits rapid light induced dehalogenation25. This may be one of the reasons behind why this dye

Figure 1.5. Phenothiazine,

xanthene and bodipy

scaffolds.

8

remains largely at the experimental in vitro stage in terms of its utility as a PS in clinical PDT20.

Addition of iodines, have also been made to BODIPY based fluorophore scaffolds (Table 1.1),

converting them into non-fluorescent highly efficient PSs with near unity singlet oxygen quantum

yields13.

Name Structure λ𝑚𝑎𝑥 (nm) Φ∆

Photofrin

Note: Actual structure is a more complex oligomer with

various ether and ester linkages.

630 0.89

Pyropheophorbide-a

665 0.45

Protoporphyrin-IX

626 0.56

9

Foscan

652 0.43

Verteporfin

686 0.76

Chlorin-e6

660 0.65

Bacteriochlorophyll-

a

770 0.35

10

Aluminum

phthalocyanine

tetrasulfonate

(AlPcS4)

676 0.38

Hypericin

590 0.43

Methylene blue

665

0.55/

0.95

(pH 9)

Toluidine Blue O

630

0.90

(pH 9)

Rose Bengal

560 0.68

11

Iodinated Bodipy

534 ~1

Table 1.1. A list of the various types of PSs in literature with their associated wavelength of photoactivation

(𝛌𝐦𝐚𝐱 ) and singlet oxygen quantum yield (Φ∆). The data presented has been obtained from [13] and [24].

Although there are numerous PSs available in literature, an ideal PS with all the desired

aforementioned traits is yet to be found. It is not surprising that the number of clinically approved

PSs remains low and therefore the utility of photodynamic therapy (PDT) has been limited.

Therefore, improvement in translational progression of PDT requires the development of novel PS

derivatives which exhibit cancer targeting behavior.

Activatable Photosensitizers (PSs)

Activatable PSs are derivatives of traditional PSs which can exist in two different states, an “on”

and “off” state13,26. In the “off” state, the excess energy endowed to the PS during the irradiation

process is taken up by a quenching molecule that is covalently attached, either directly or via a

linker scaffold, to the PS13,26. This quenching effect prevents the occurrence of intersystem

crossing, abolishing the singlet oxygen production by the PS13,26. In other words, the “off” state

PS cannot induce cell death even upon irradiation (Figure 1.6). On the other hand, when in the

“on” state, the PS is structurally modified such that the quencher molecule is no longer covalently

bound, allowing for singlet oxygen production13,26. Put simply, irradiation of the “on” state PS

leads to cell death (Figure 1.6). This “off/on” phenomenon can be exploited to achieve cancer

targeting behavior by linking the activation, that is the transition from the “off” to “on” state due

to quencher PS separation, by the action of a cancer biomarker (Figure 1.6). By connecting the

activation of the PS to the action of small molecules or enzymes that are more abundant/active in

cancer cells relative to their healthy counter parts, more PS will be activated in the cancer cells

compared to the healthy cells. This will translate to greater phototoxicity in cancer cells with

minimal, if any, toxicity in healthy cells.

12

A key component in the development of successful activatable PSs is the use of the appropriate

quenching mechanisms to ensure the PS exhibits minimal singlet oxygen production. In the next

section, a brief survey of the different mechanisms of PS quenching and their application in

development of novel activatable PSs are presented.

Figure 1.6. A schematic of the mechanism of action of an activatable PS. In the off state, the PS cannot produce

singlet oxygen upon irradiation and hence the viability of the cells is not impacted. In the presence of a cancer

biomarker, the PS is activated (turned on) and therefore irradiation of the cells results in production of singlet

oxygen and thereafter cell death.

Mechanisms of quenching & activatable photosensitizers

The generation of singlet oxygen by a PS must proceed via the triplet excited state which in turn

is generated by the electronic rearrangement of the singlet excited state13,26. This excited state

electronic rearrangement occurs via a photophysical process called intersystem crossing (ISC)13,26.

Therefore, photophysical processes which compete and thereby prevent ISC can render the loss of

photosensitization13,26. Such processes include, Förster resonance energy transfer (FRET),

photoinduced electron transfer (PeT), and intramolecular charge transfer (ICT)13,26. In addition to

the photophysical processes, there are also cap and release approaches which employ caged groups

to trap the PS in a non-active form, followed by the subsequent release which results in activation26.

Commonly employed in the field of fluorescent sensors, FRET refers to a photophysical

phenomenon in which the light emitted, called fluorescence, by a donor molecule is transmitted to

an acceptor molecule in close vicinity (less than 10 nm)13,26,27. The acceptor molecule then goes

on to emit the energy in the form of longer wavelength fluorescence. In other words, excitation of

the donor molecule results in emission by the acceptor molecule (Figure 1.7)13,26,27. However, the

acceptor molecule does not always have to be emissive. In some cases, like for activatable PSs, a

non-emissive dark FRET quencher may be utilized13,26–28. These acceptors dyes are very efficient

13

at undergoing internal conversion and therefore release the energy in the form of heat rather than

fluoresence13,26,27. If a PS is near a FRET quencher which absorbs at the emission wavelength of

the PS, the PS cannot undergo ISC and produce singlet oxygen/fluoresence13,26.

Figure 1.7. FRET is based upon a non-radiative transfer of energy from a donor (“D”) fluorophore to an

acceptor (“A”). Excitation of the donor with appropriate wavelength leads to the emission by the acceptor via

some long wavelength fluorescence. This image was obtained from [26] and [27].

This combination of PS and FRET quencher can be utilized to develop an “off” state PS. Since the

FRET quenching effect is strongly dependent on the distance between the FRET pair (PS and

quencher), separation of the two scaffolds results in activation or turning on of the PS13,26–29. By

utilizing the appropriate FRET pair and coupling the separation of the two moieties with the action

of a cancer biomarker, an activatable PS can be prepared. In fact, as shown in Figure 1.8, this

strategy has been employed for the development of protease activatable PSs, where a known

peptide substrate for a protease overexpressed in cancer is modified with a PS (such as

14

pyropheophorbide a) and the appropriate dark quencher (blackhole quencher 3)13,26,29. As

discussed, the PS in this state is unable to produce singlet oxygen. However, upon enzymatic

cleavage of the peptide substrate, the PS and the quencher are separated, leading to liberation of

the active PS capable of producing singlet oxygen and fluoresence13,26,29.

Figure 1.8. A schematic of the mechanism of activation of protease based activatable PSs based on principles

of FRET. The modification of a peptide substrate with a PS and the appropriate non-emissive FRET acceptor

(quencher, “Q”), leads to an off state where singlet oxygen and fluorescence is not produced. Upon enzymatic

cleavage of the peptide, the PS (Donor, “D”) is separated from the FRET quencher, leading to activation and

restoring fluorescence and singlet oxygen production. This image was obtained from [29].

The second photophysical process that can be utilized in quenching applications is photoinduced

electron transfer (PeT). As shown in Figure 1.9, PeT is an electron transfer or redox reaction that

occurs in the excited state26,30. Irradiation of the PS (labelled fluorophore in Figure 1.9) induces an

electronic excitation, moving the electron from the highest occupied molecular orbital (HOMO)

to the lowest unoccupied molecular orbital (LUMO)26,30,31. Normally, this electron will then relax

back to the ground state, emitting fluoresence26,30,31. Alternatively, the PS may also undergo ISC

to the triplet excited state and subsequently produce singlet oxygen26. However, in the presence of

an electron donor, the excess excitation energy brought on by the vacancy in the HOMO of the

PS, can be quenched by the donor species via an electron transfer to the fill the vacancy26,30,31. This

reduction of the PS results in the loss of fluorescence and photosensation properties26,30,31. Note,

the process is fully reversible such that the excited electron is often recovered by the oxidized

electron donor, returning the entire system to the ground state26,30,31.

15

Figure 1.9. A schematic overview of the quenching due to photoinduced electron transfer (PeT). The electron

transfer by the donor group (quencher) to the vacancy produced by photoexcitation of PS in the HOMO serves

to quench the excited state. This image was obtained from [31].

Much like FRET, which requires proximity and spectral overlap between the FRET pair, PeT also

has certain requirements27,28. The redox potentials of the electron donor moiety and the desired PS

must be compatible to allow the electron transfer in the excited state30. In addition, to achieve the

electron transfer, the electronic wave functions of the species must overlap and therefore, the donor

and acceptor moieties must be close together30. Consequently, PeT generally requires much shorter

distances in comparison to FRET. Nonetheless, PeT has been quite useful in the development of

activatable PSs, for example in the case of iodinated BODIPY dyes with aniline moieties32. As

shown in Figure 1.10, in the neutral state, the amine in the aniline moiety is capable of donating

electrons to the BODIPY PS32. This PeT quenching effect inhibits ISC and singlet oxygen

production32. However upon protonation of the amines, the electrons are no longer available for

donation, eliminating the PeT quenching, thereby allowing ISC and production of singlet oxygen32.

In this scenario, the pH dependent protonation of the aniline allows for the modulation of the PeT

quenching effect and as such can be exploited for the development of pH-dependent activatable

PSs.

The third photophysical phenomenon which may be employed in quenching of PSs is

intramolecular charge transfer (ICT)26,30. ICT is also an electron transfer process, however, unlike

PeT which utilizes spatially separated acceptor and donors, ICT occurs within the same system

(i.e. among two different functional groups) or within two systems that are in direct conjugation

with each other26,30. An example of this approach is shown in Figure 1.11. Here, a porphyrin-based

PS is conjugated to an anthracene which acts as an ICT based quencher for the PS, minimizing

singlet oxygen production26,33. This charge transfer induced quenching effect is eliminated upon

16

binding of the positively charged porphyrin to negatively charged DNA, restoring the singlet

oxygen production26,33.

Figure 1.10. A schematic of the PeT based quenching of a pH dependent, BODIPY based activatable PS. In the

neutral state, the amine in the aniline moiety serves as an electron donor and therefore quenches the singlet

oxygen production by the BODIPY PS. Upon protonation of the amine, the electron transfer is eliminated,

abolishing the PeT quenching and restoring the singlet oxygen production. This image was obtained from [32].

Figure 1.11. A schematic of the activation of the ICT quenched porphyrin based activatable PS. The anthracene

moiety directly conjugated to the cationic porphyrin serves as a charge donor, quenching the ISC of the

porphyrin and thereby inhibiting singlet oxygen production. The binding of the positively charged porphyrin

to a piece of negatively charged DNA, diminishes the charge transfer between the anthracene and PS, restoring

singlet oxygen production. This image was obtained from [33].

17

The last approach which can be used to develop activatable PSs is based upon caging or trapping

of the desired PS in a non-active form with molecular scaffolds called cages26. Upon enzymatic

removal of such cages, the active PS is liberated. This strategy has been quite actively employed

in the development of various enzyme activatable PSs utilizing methylene blue34,35. As shown in

Figure 1.12, this approach is based upon caging methylene blue (MB) in a reduced non-active form

called leuco methylene blue (LMB)34,35. In this form, the aromaticity of the PS is interrupted such

that chromophore is no longer able to produce singlet oxygen or fluorescence34,35. Upon enzymatic

removal of the caged scaffold, LMB is released which undergoes rapid spontaneous oxidization

due to the surrounding the environment, producing photoactive MB34,35.

Figure 1.12. A schematic of the caged approach to developing Methylene blue (MB) activatable PSs. The

reduced non-active form of MB called leuco MB is trapped with an enzyme cleavable molecular scaffold or

“cage”. Upon enzymatic removal of the cage, Leuco MB is released which rapidly undergoes spontaneous

oxidation to the photoactive MB.

Clearly there are numerous methods by which the photophysical properties of chromophores may

be modified such that they cannot produce fluorescence and/or singlet oxygen. Unfortunately, as

demonstrated by the existence of only a handful activatable PSs, the concept of enzyme activatable

PSs is yet to be truly embraced in the field of PDT13,26. In addition, the activatable PSs published

in literature have several drawbacks. For example, the FRET based activatable PSs are centered

around peptide substrates, which are known to have poor cellular permeability and therefore have

limited application in vivo13,26. On the other hand, current PeT based activatable PSs are largely

activated in a pH dependent manner rather than enzymes overexpressed in cancer26,32. Much like

the PeT probes, the ICT based activatable PSs are also at a proof of concept level, with no

connection with enzyme activatability26,33. Therefore, the development of novel small molecule-

based enzyme activatable PSs which may surpass such limitations is highly warranted. In this

regard, the next two chapters will discuss the on-going development of two novel enzyme

activatable PS probes. The greater cancer selectivity of the enzyme activatable PSs is expected to

produce minimal if any side effects and therefore increase the clinical utility of PDT.

18

Chapter 2 On the road to developing an MGMT activatable Photosensitizer

O6-Methylguanine DNA Methyltransferase (MGMT) as a PDT target

It is well known that the instructions needed for the survival and proper functioning of living

organisms are stored in a library of biomolecules called DNA. Sometimes this compendium of

biomolecules can become damaged, leading to a loss of information and proper functioning in the

organism, manifesting in the form of diseased states. Such damage can be brought on by alkylation

due to endogenous alkylating agents such as S-adenosylmethionine or exogenous sources such as

tobacco smoke and UV light36–38. The induced alkylation can lead to point mutations which may

serve to activate or suppress cancer related genes or alternatively produce double stranded breaks

in DNA with subsequent apoptotic cell death36–39. One site of alkylation that is especially

mutagenic is the O6 position of guanine, which is known to cause GC to AT mutations and if not

appropriately repaired, induce apoptosis36–39. To cope with such damage, nature has developed

several different repair pathways. One such pathway, is the direct repair pathway which includes

a small DNA repair enzyme (207 amino acid) called O6-methylguanine DNA methyltransferase or

MGMT (E.C. 2.1.1.63) (Figure 2.1)36–38,40.

Figure 2.1. A crystal structure of Human MGMT (E.C. 2.1.1.63). This Figure was obtained from [40].

MGMT is responsible for the repair of methylation at the mutagenic O6 position of guanine in a

single irreversible step37,38,41. Although MGMT shows a preference for O6 methylated guanine,

larger alkyl groups such as ethyl and butyl can also be repaired, albeit less efficiently36–38. It is

worth nothing that MGMT can repair DNA in both condensed and open chromatin forms, working

19

independently without an assortment of proteins/cofactors, characteristic of other DNA repair

pathways such as mismatch repair (MMR)38. To repair the lesions, the alkylated guanine is brought

into the active site of the MGMT, where the methyl group is transferred to a cysteine residue

(cys145 in humans) in an acid catalyzed SN2 like fashion, with final covalent modification of the

enzyme36–38,42. The transfer process is thought to be facilitated by a network of hydrogen bonding

involving water, histidine, glutamic-acid, asparagine, and tyrosine residues in the active site,

increasing the nucleophilicity of the thiolate and the electrophilicity of the alkyl group at O6

position (Figure 2.2)38,42. The resultant alkylated protein is then ubiquitinated and subsequently

degraded by the proteasomal machinary36–38. Since MGMT does not turnover, the repair of O6

adducts is highly dependent on the amount and rate of MGMT produced by the cell36–38.

Figure 2.2. MGMT is a DNA repair enzyme responsible for removal of alkylations at the O6 position of guanine.

The protein utilizes a cysteine residue at its active site as a nucleophile in combination with a hydrogen bonding

network made up of water and several amino acid residues (Tyr, His, Asn, Glu). The alkyl group is transferred

to the cysteine permanently alkylating the protein and restoring native guanine. These images were obtained

from [38] and [42].

Although beneficial in the case of erroneous alkylation of the DNA, the activity of this ubiquitous

enzyme can also serve as a big hinderance in the potency of chemotherapeutics36–38. Many anti-

cancer chemo agents such Temozolomide (TMZ) exert their cytotoxic effect via alkylation at O6

position of guanine43. This is problematic, as the action of MGMT can simply repair these lesions

canceling any potential cytotoxicity. In other words, the action of MGMT serves as a mechanism

of chemo resistance lowering the efficacy of alkylating drugs for cancer treatment36–38,43. This

20

situation is further worsened by the fact that MGMT is overexpressed in several different types of

cancers (Figure 2.3)41.

Figure 2.3. MGMT is a ubiquitous enzyme found in all tissue. It is overexpressed in several different types of

cancers such as breast, brain, colon, lung, rectum and stomach and underexpressed in other such as blood, liver

and testis. This image was obtained from [41].

To resolve this issue, small molecule

inhibitors to block the MGMT mediated

repair have been developed. Two of the most

well-known MGMT inhibitors are O6-benzyl

guanine (O6BG) and O6-(4-bromothenyl)

guanine (Patrin-2) with IC50 values of 200

nM and 4 nM respectively (Figure 2.4)44.

Derivatives of O6 alkylated guanine base,

O6BG and Patrin-2 function as

pseudosubstrates for the enzyme, that is, the

enzyme will transfer the alkyl group at the

O6 position - benzyl for O6BG and 4-

bromothenyl for Patrin-2 - on to itself, losing

function (Figure 2.4)44. O6-BG and Patrin-2

Figure 2.4. O6-BG and Patrin-2 are pseudosubstrates for

MGMT. They act as structural mimics of alkylated

guanine and therefore are repaired by MGMT, resulting

in enzyme alkylation and thereafter inactivation.

21

have been clinically tested and found to induce minimal toxicity when administrated alone45,46.

The established overexpression of MGMT and its mechanism of action

in combination with the availability of small molecule inhibitors, makes

MGMT an attractive target for exploitation in the development of

activatable photosensitizers (PSs). It is well known that addition of

substituents at the para position of the benzyl group and the solvent

exposed N9 and C8 positions of the guanine base in O6 BG are well

tolerated by the enzyme(Figure 2.5)40,47–50. Combining this information

with the mechanism of action of MGMT, O6-BG can be modified with a

PS at the para position of the benzyl group and the appropriate FRET

quencher at the N9 position of the guanine base to develop a FRET

quenched “off” state PS (Figure 2.6). Upon action of MGMT, the PS

conjugated benzyl group will be transferred to the enzyme active site,

thereby separating it from the quencher on the guanine base, eliminating

the FRET quenching effect, thereby turning on the PS. With this design

in mind, probe 1 or P1 was synthesized (Figure 2.7).

Figure 2.6. Modification of O6-BG with a PS at the para position of the benzyl group and the appropriate

FRET quencher at the N9 position allows for the development of a FRET quenched “Off” state PS. Upon action

of MGMT, the benzyl group with the PS is transferred onto the active site of the enzyme resulting in separation

from the quencher group, eliminating FRET quenching and activating/ turning “on” the PS.

Figure 2.5.

Modifications of O6-

BG at the indicated

positions are well

tolerated by MGMT.

22

Figure 2.7. Structure of P1 (probe 1). The commercially available PS, Rose Bengal (λem 575 nm), is utilized in

combination with the FRET quencher black hole quencher 2 (BHQ2) (λabs 560-670 nm).

As shown in Figure 2.7, P1 contains Rose Bengal (λem 575 nm) as the PS and black hole quencher

2 (BHQ2) (λabs 560-670 nm) as the dark FRET quencher13. Rose Bengal was selected due to its

commercial availability, inexpensiveness, good fluorescence and singlet oxygen quantum yields13.

Meanwhile, BHQ2 was selected as its absorption spectrum overlaps with the emission spectrum

of Rose Bengal13.

Results and discussion

Probe 1 (P1) was prepared as shown in Figure 2.8. Rose Bengal (RB) was modified at the carboxyl

position with a 3-azido-N-methylpropan-1-amine linker using HATU mediated amine carboxyl

acid coupling, to obtain RB with an azide functional handle (RB-N3). Next, free guanine base

modified with an O6 benzyl group with an alkyne handle at the para position (compound 2) was

prepared. This scaffold was subsequently modified at the N9 position with the commercially

available propylamine linker to obtain compound 3. The resultant amine handle was utilized to

couple the prepared scaffold (compound 3) with BHQ2-NHS to obtain an alkyne modified O6-

Benzyl guanine with a quencher at the N9 position (compound 4). This scaffold was then finally

23

coupled to the azide functionalized RB PS using copper catalyzed click chemistry to obtain P1

(compound 8). For further details on the synthesis, refer to the materials and methods section

below.

As shown in Figures 2.9-2.11, the λmax, λex, and λem were found to be 565, 568, and 577 nm,

respectively, matching closely with literature13. The probe was then investigated for activatable

behavior using a fluorescence-based assay. As shown in Figure 2.12, in the synthesized form of

P1, the proximity of the FRET quencher (BHQ2) to the fluorescent PS (RB) should yield low

fluorescence. Upon action of MGMT, RB is expected to transfer onto the enzyme, separating it

from the quencher, thereby leading to an increase in fluorescence emission (Figure 2.12).

However, as shown in Figure 2.13, incubation of P1 (58 nM) with MGMT (500 nM) yielded no

change in fluorescence, suggested that P1 was not activated by MGMT.

Figure 2.9. UV-Visble spectrum of P1 (6.3 𝝁M) in basic ethanol. The compound was found to have λmax at

565 nm.

0

0.1

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

400 450 500 550 600 650 700

Ab

sorb

ance

Wavelength (nm)

Absorption Spectra

λ max – 565 nm

24

Figure 2.8. The synthetic scheme for the synthesis of probe 1 (P1) labelled as “8”.

25

Figure 2.10. Fluorescence excitation spectra of P1 (58 nM) in buffer containing 70 mM HEPES (pH 7.8), 5mM

EDTA, 1 mM DTT and 1% (v/v) DMSO. The compound was found to have λex at 568 nm.

Figure 2.11 Fluorescence emission spectra of P1 (58 nM) in buffer containing 70 mM HEPES (pH 7.8), 5mM

EDTA, 1 mM DTT and 1% (v/v) DMSO. The compound was found to have λem at 577 nm.

26

Figure 2.12. A schematic of the mechanism of activation of P1 by MGMT.

Figure 2.13. The obtained fluorescence intensity as a function of time for P1 (58 nM) with MGMT (500 nM) in

70 mM HEPES (pH 7.8), 5mM EDTA, 1 mM DTT and 50 µg/mL BSA. The obtained horizontal curve indicated

a lack of activation.

To understand which feature of the probe (the PS or the quencher) was responsible for the lack of

recognition by MGMT, an always-on variant of P1, called P1NQ (Figure 2.15), containing no

modifications at the N9 position of guanine was designed. The choice of PS in P1NQ was revised

such that Pyropheophorbide-a (“Pyro”) would be utilized instead of RB. This revision was

proposed due to the reported poor photostability of RB25, and so, the use of a clinically approved

photostable porphyrin-based PS was adopted. The synthesis of P1NQ was initiated with the

0

200

400

600

800

1000

1200

1400

1600

1800

0 200 400 600 800 1000 1200

Flu

or.

Inte

nsi

ty

Time (sec)

27

modification of the carboxylic acid handle of Pyro with a sarcosine linker (compound 12) (Figure

2.16). The modified pyro was then coupled to the 4-methylamine-O6 benzyl guanine scaffold

(compound 11), to obtain the desired probe (Figure 2.16). Details of synthetic procedures are

provided in the materials and methods section below.

Figure 2.15. The chemical structure of probe 1 no quencher (P1NQ).

Once synthesized, the probe was examined for its ability to covalently label the active site of

MGMT. Since P1NQ is not an activatable probe, an SDS PAGE assay was used to determine if

covalent transfer occurred. In this assay, the enzyme (1 µM) was incubated with the probe (2 µM)

at 37oC overnight, in the dark. The mixture was then combined with an SDS and β-

Mercaptoethanol (BME) containing loading buffer and boiled (90oC) for 10 minutes to induce

denaturation. The resultant mixture was then run through a SDS polyacrylamide gel via

electrophoresis, separating the contents based on their molecular weight. The electrophoresed gel

was then exposed to blue light and the fluorescent response (red light) from Pyro was measured.

If the MGMT was tagged, a fluorescent band matching the molecular weight of the MGMT plus

the transferred group (24.2 kDa) should be observed. As shown in Figure 2.17, this was indeed

the case, indicating the successful transfer of the benzyl group with the sarcosine modified Pyro

to the MGMT active site. To further confirm that the hypothesized tagged MGMT band is indeed

a protein, silver straining was performed resulting in a single band characteristic of the desired

species (Figure 2.17).

28

Figure 2.16. (Top) The synthetic scheme for the synthesis of the necessary precursor molecules for the synthesis

of P1NQ. (Bottom) The synthetic scheme for the synthesis of P1NQ.

29

Figure 2.17. SDS PAGE assay for the investigation of PS transfer to the active site of MGMT. The sample was

prepared via addition of 1 µM MGMT to 2 µM P1NQ in 10 mM PBS (pH 7.4) and 1mM DTT, incubated for

16 hours in the dark at 37oC. The electrophoresed gel was excited with blue light and fluorescent emission was

collected in the red region. The fluorescent image of the gel exhibited two bands, one characteristic of the

combined mass of MGMT and PS (24.2 kDa) and the other characteristic of the free probe (unlabeled). The

presence of protein was confirmed via subsequent silver staining of the gel.

Based on the observed results, MGMT can act upon P1NQ and covalently transfer the desired PS

on to its active site. Unfortunately, a large portion of the probe (“free dye”) was found to unlabeled,

indicating a poor efficiency of transfer (Figure 2.17). To investigate this, another fluorescence SDS

page assay was performed with several different concentrations of P1NQ and MGMT, alongside

controls with only MGMT or P1NQ, and a sample with MGMT pre-treated with inhibitor (O6BG)

(Figure 2.18). As expected, lane A with only MGMT (no P1NQ) showed no fluorescent bands,

while lane F, with only P1NQ (no MGMT), showed only a single fluorescent band characteristic

of the free probe. As previously observed, lanes B-E with both MGMT and P1NQ, exhibited two

fluorescent bands characteristic of tagged MGMT and free P1NQ. In addition, the fluorescence

intensity of the tagged MGMT in these lanes was found to increase as a function of the combined

concentrations of MGMT and P1NQ as indicated by the relative fluorescence intensity (Ir) of the

bands in lane C (0.17) and lane D (0.75) relative to lane D (1.00) (Figure 2.18). Furthermore, the

incubation of MGMT with O6BG prior to the addition of P1NQ (lane B) was found to decrease

the fluorescence intensity of the tagged MGMT band (Ir 0.35 relative to lane E), indicating that

P1NQ is covalently transferred to the active site of the enzyme.

30

Considering the promising results from the fluorescence SDS page assay, in cellulo labeling of

MGMT was examined. As shown in Figure 2.19, P1NQ was added to 2 different cell lines, breast

cancer MCF7 cells with overexpressed MGMT, and MGMT deficient human embryonic kidney

cells, HEK293T. Post incubation and wash, it was expected that the MCF7 cells would exhibit

bright red fluorescence representative of in cellulo MGMT labelling, while the HEK293T cells

would exhibit very minimal or no fluorescence indicating a lack of labeling. Unfortunately, as

shown in Figure 2.19, there was no significant difference in fluorescence in the two cell lines. The

lack of difference was attributed to two factors: i) the hydrophobic nature of pyro and ii) poor

labeling efficiency of MGMT by P1NQ. The hydrophobic nature of pyro was believed to induce

aggregation of the PS and non-specific binding to cellular components upon internalization into

the cell. This non-specific binding effect is hypothesized to impede the adequate washout of P1NQ,

producing significant background fluorescence, leading to similarity in the fluorescent images of

the two cell lines. This situation is further worsened by the poor labeling efficiency of MGMT by

P1NQ, as observed in the fluorescence SDS PAGE assay. This poor labeling may translate to

F E D

C B

A

Tagged

MGMT

Figure 2.18. Fluorescence SDS PAGE assay. Lane A: 1 µM MGMT, lane B: 1 µM MGMT, 0.05 µM O6BG, 1

µM P1NQ, lane C: 0.25 µM MGMT and P1NQ, lane D: 0.5 µM MGMT and P1NQ, lane E: 1 µM MGMT and

P1NQ, lane F: 1 µM P1NQ. Refer to the materials and methods for details.

Free

P1NQ

31

insufficient tagging of the MGMT in MCF7 cells and therefore, the fluorescence signal may not

be as intense as would be expected if the labeling was more efficient. Consequently, the

background signals (fluorescence in HEK293T cells) and the labelled MGMT in MCF7 may be

indistinguishable. To resolve such limitations of P1NQ and ultimately produce a potent activatable

variant, several future modifications are suggested in the section below.

Figure 2.19. Bright field (top) and fluorescent images (bottom) of MCF7 cells (left) with overexpressed MGMT

and HEK293T cells (right) with no MGMT, post 3-hour incubation with 0.85 µM P1NQ and subsequent wash

with 0.01 M PBS (pH 7.4) buffer. Images were taken with a 20X magnification.

Future directions & experiments

As established in the work above, P1NQ is a Pyropheophorbide-a based PS probe, which exhibits

the ability to covalent label native MGMT; a cancer biomarker. As such, P1NQ provides a

foundation from which one may begin the development of a FRET-based MGMT activatable PS

probe. Before one may install a quencher group, it is worth addressing and resolving the limitations

exhibited by P1NQ. It is worth considering the role of linker lengths between the PS and the

32

MGMT recognition scaffold (O6-benzyl guanine), to examine the role of such effects on labeling

efficiency. By utilizing longer linkers, one may potentially decrease any steric effects at the active

site of the enzyme, and thereby improve the labeling efficiency. In addition, it is also worthwhile

to explore the use of different PSs, such that the issue of non-specific binding due to

hydrophobicity of the PS may be eliminated. These two routes may yield a P1NQ derivative, which

could be utilized in washout type assays and serve as a starting point for introducing a quencher

group. Regarding the development of the activatable variant, the length of the linker from the

MGMT recognition scaffold to the quencher should also be investigated. Finally, if the probe is

found to exhibit poor activatability even after all the above-mentioned optimization, then

derivatization of a more potent MGMT inhibitor (e.g. PaTrin-2) should be explored.

Figure 2.20. A schematic of the potential future directions to develop a potent MGMT activatable PS probe.

In addition to developing FRET-based MGMT activatable PS probes, the utilization of the PeT

quenching abilities of guanine base may also be exploited to develop PeT based activatable PS

probes, where no extra quencher molecule is necessary51. To develop such probes, O6BG scaffolds

can be conjugated to a potential PeT compatible PS such as the coumarin based 3AIC (see chapter

four for details on 3AIC) (Figure 2.21)52. The proximity of such PSs to the guanine base may result

in a PeT quenched “off” state of the PS. Once the PS has been separated by the action of MGMT,

the PeT quenching effect may be eliminated yielding an activated PS.

33

Figure 2.21. Proposed structure of a PeT based MGMT activatable PS probe (O6BG-PL-3AIC) using a novel

PS, 3AIC (see chapter four for details on 3AIC).

Materials & methods

General Methods

All reactions were conducted in oven-dried glassware under atmospheric conditions, without the

use of inert gas protection. All reagents for which the synthesis is not provided were purchased

from Sigma Aldrich or Alfa Aesar and used as purchased without further purification. NMR

solvents were purchased from Cambridge isotope laboratories and used without further

purification. Final probe solutions were prepared using high grade DMSO from Thermo Fisher

Scientific. PBS pH 7.4 (1X) buffer was purchased from Thermo Fisher Scientific. NMR data was

collected using a Bruker Avance III 400 MHz spectrometer, with chemical shifts reported in ppm

relative to the residual solvent resonance peaks. Fluorescent spectra were obtained using a

Shimadzu RF-6000 Spectro fluorophotometer while UV-Vis spectra were obtained using a

Shimadzu UV-1800. Fluorescent microscopy was performed using an Olympus IX73. Gel

electrophoresis was performed using the Bio-Rad Mini-PROTEAN® electrophoresis system.

MGMT was purchased from Creative Biomart. All cells were maintained in an atmosphere of

5/95% (v/v) of CO2/air at 37oC. MCF-7 and HEK293T cells were grown in Dulbecco’s

Modification of Eagle’s Medium (DMEM (1X)/high: with 4.5 g/L Glucose, L-Glutamine, without

sodium pyruvate) supplemented with 10% fetal bovine serum, 100 units/mL penicillin, and 100

μg/mL streptomycin. All cells were plated in 8-chambered wells (Lab-Tek® Chambered #1.0

Borosilicate Coverglass System) at a density of 5×104cells/well one day before imaging.

34

UV-Vis Spectroscopy

Using a 60 𝜇L quartz cuvette, the UV-Vis spectrophotometer was blanked with basic ethanol.

Then, 0.6 𝜇L of a 0.63 mM P1 stock solution (in DMSO) was added to 59.4 𝜇L basic ethanol to

obtain a UV-Vis spectrum of P1 (final concentration 6.3 𝜇M). Note, concentration was determined

using the extinction coefficient (90,000 M-1cm-1) for Rose Bengal in ethanol as reported in

literature13. The procedure was then repeated with a stock solution of P1NQ, using PBS (pH 7.4)

instead of basic ethanol.

Fluorescence Spectroscopy

Excitation spectrum: A sample of P1 (58 nM) at 37oC in buffer containing 70 mM HEPES (pH

7.8), 5mM EDTA, 1 mM DTT and 1% (v/v) DMSO, was excited at various wavelengths (250 to

570 nm) and the resultant fluorescent emission at 577 nm was measured. Note, 3 nm excitation

and emission wavelengths were utilized.

Emission Spectrum: A sample of P1 (58 nM) at 37oC in buffer containing 70 mM HEPES (pH

7.8), 5mM EDTA, 1 mM DTT and 1% (v/v) DMSO, was excited at 568 nm and the fluorescent

emission was measured in the 577-640 nm (Figure 2.11). Note, 3 nm excitation and emission

wavelengths were utilized.

Fluorescence Activatability Assay: A sample of P1 (58 nM) at 37oC in buffer containing 70 mM

HEPES (pH 7.8), 5mM EDTA, 1 mM DTT and 1% (v/v) DMSO was excited at 568 nm and the

resultant fluorescent emission at 577 nm was measured as a function of time. After 5 minutes of

incubation, MGMT (0.6 μM) was added to the sample and the fluoresence intensity was monitored.

In cases where no change was observed, the measurements were performed for atleast 20 minutes.

Note, 3 nm excitation and emission wavelengths were utilized.

SDS PAGE

Casting SDS-PAGE Gels: The separating gel was prepared as follows. 4.1 mL DDI water, 3.3

mL acrylamide (37.5%), 2.5 mL Tris-HCl (1.5 M, pH 8.8), 0.10 mL SDS (10% by volume) were

added to a 50 mL falcon tube and the mixture was briefly vortexed. Then, 0.01 mL TEMED

followed by 0.03 mL APS (10%) was added to the falcon tube and the mixture was briefly

vortexed. The contents were uniformly poured between two glass spacer plates clamped onto the

35

Mini-PROTEAN® tetra cell casting stand, until the solution volume was about 1 cm from the top.

The apparatus was allowed to sit for 30 minutes to allow solidification of the gel matrix. In the

meantime, the stacking gel was prepared as follows. 6.1 mL DDI water, 1.3 mL acrylamide

(37.5%), 2.5 mL Tris-HCl (0.5 M, pH 6.8), 0.10 mL SDS (10% by volume) were added to a 50

mL falcon tube and the mixture was briefly vortexed. Then, 0.01 mL TEMED followed by 0.01

mL APS (10%) was added to the falcon tube and the mixture was briefly vortexed. Once the 30-

minute incubation time was complete, the stacking gel matrix was poured between the two glass

spacer plates until the solvent reached the top of the plates. Then, the comb was carefully inserted

between the spacer plates and gel was allowed to solidify for 30 minutes. Upon solidification, the

gel plates were wrapped in damp paper towels and stored at 4oC for up to 1 week.

The Assay: 10 uL samples were prepared in 1.5 mL centrifuge tubes with the appropriate

components for each lane (see table below). The sample tubes were wrapped in foil and incubated

in a 37oC water bath for overnight. Note, for the inhibitor lane, MGMT and O6BG were first

incubated for 2 hours prior to addition of P1NQ. Then, the tubes were briefly centrifuged and 10.0

uL 2X SDS loading buffer (62.5 mM Tris-HCl pH 6.8, 25% glycerol and 2% SDS, 5% BME) was

added to each sample tube. The samples were then incubated at 90oC for 10 minutes and 10 uL of

each sample was loaded on to the SDS-PAGE gel. Note, 7.5 uL of the ladder was used. The gel

electrophoresis was run at a constant 250 volts for about 30 minutes. The gel was then imaged via

excitation in the blue region and the emission measurement in the red region.

Lane Component (Amounts)

A DTT (1 mM), MGMT (1 µM), PBS

B

DTT (1 mM), MGMT (1 µM), O6BG (50 µM) PBS – 2 hours at 37oC. Then P1NQ (1

µM)

C DTT (1 mM), MGMT (250 nM), P1NQ (250 nM), PBS

D DTT (1 mM), MGMT (500 nM), P1NQ (500 nM), PBS

E DTT (1 mM), MGMT (1 µM), P1NQ (1 µM), PBS

F DTT (1 mM), P1NQ (1 µM), PBS

Refer to Figure 2.18 for the gel.

36

In cellulo labeling

Cells were incubated with 0.85 𝜇M P1NQ for 3 hours in Opti-MEM. They were then washed

with 250 uL PBS 3 times before being imaged in OPTI-MEM.

Synthesis (Refer to Figure 2.8 and 2.16 for full schemes)

Guanine Salt (1): Guanine salt was synthesized based on literature53 using DMF instead of DMSO

as the solvent. Under argon, 1.30 grams (7.6 mmol) of 2-Amino-6-chloropurine (chloroguanine)

was added to 40 mL anhydrous DMF and stirred at 40oC for 30 minutes. The obtained clear yellow

solution was allowed to sit at room temperature for 10 minutes. Then, 5 mL (47 mmol) of 1-

methylpyrrolidine was added and the mixture was allowed to stir at room temperature for 3 days.

After 3 days, a yellow solution with a white precipitate was obtained. 6 mL acetone was added to

the solution to complete the precipitation. The precipitate was isolated via vacuum filtration,

washed with acetone and air dried to obtain the desired product as a white solid (1.67 g (6.55

mmol), yield 86.8%). 1HNMR (400 MHz DMSO-d6): 2.06 (m, 2H), 2.25 (m, 2H), 3.64 (s, 3H),

3.94 (m, 2H), 4.59 (m, 2H), 7.11 (s, 2H), 8.34 (s, 1H), 13.30 (bs, 1H). See appendix A for the

spectra.

37

Alkyne O6 Benzyl Guanine (2): Under argon, 302 mg (2.2 mmol) of 4-ethynylbenzyl alcohol was

added to 9 mL anhydrous DMF and stirred at room temperature for 2 minutes to obtain a clear

yellow solution. Then, 514 mg (4.4 mmol) of potassium tert-butoxide was added, resulting in a

dark red/maroon solution. Next, 300 mg (1.2 mmol) of guanine salt (1) was added, and the mixture

was stirred at room temperature for 3 hours. The solvent was removed in vacuo. The sample was

then dry loaded on to a column and further purified via flash chromatography (100%

dichloromethane to 95% dichloromethane with 5% methanol) to obtain the desired product as a

yellow solid (237 mg (0.893 mmol), yield 75.7%). 1HNMR (400 MHz DMSO-d6): 4.20 (s, 1H),

5.50 (s, 2H), 6.30 (s, 2H), 7.50 (s, 4H), 7.81 (s, 1H), 12.43 (s, 1H). See appendix A for the spectra.

Alkyne O6 Benzyl Guanine N9 Propylamine (3): Under argon, 17 mg (0.06 mmol) of alkyne O6

benzyl guanine (2) was added to 2.65 mL anhydrous DMF and stirred at room temperature for 5

minutes. Then, 4 mg (0.1 mmol) of sodium hydride (60 % dispersion in mineral oil) was added to

the clear yellow solution to obtained orange/red solution. The mixture was stirred for about 5

minutes and then 14 mg (0.06 mmol) of 3-bromopropylamine hydrobromide was added. The

solution was stirred at room temperature for 3 hours, while monitoring the reaction with TLC (90%

ethyl acetate with 10% methanol) and ninhydrin staining. After 3 hours, the reactant was fully

consumed, and the reaction was assumed to be completed. The crude yellow oil was used without

any further purification. See appendix A for the spectra.

38

Alkyne O6 Benzyl Guanine N9 Propylamine BHQ2 (4): Under argon, 3 mg (0.006 mmol) of

blackhole quencher NHS (BHQ2-NHS) was added to 150 𝜇L anhydrous DMF and stirred room at

temperature for 2 minutes. Then, 1.8 mg (0.006 mmol) of Alkyne O6 Benzyl Guanine N9

Propylamine (3) and 4 𝜇L (0.03 mmol) of triethylamine were added to the purple solution. The

reaction was stirred for 24 hours at 40oC. The solvent was removed in vacuo and the obtained

crude product was purified via flash chromatography (100% ethyl acetate to 80% ethyl acetate

with 20% methanol) to obtain the desired product as a purple solid (2.90 mg (0.005 mmol), yield

65.5%). 1HNMR (400 MHz DMSO-d6): 1.81 (m, 2H), 1.89 (m, 2H), 2.18 (m, 2H), 2.68 (m, 2H),

3.07 (s, 6H), 3.95 (s, 4H), 4.01 (s, 6H), 4.20 (s, 1H), 5.50 (s, 2H), 6.46 (s, 2H), 6.89 (d, 2H, J =

9.35 Hz), 7.39 (s, 1H), 7.46 (s, 1H), 7.50 (s, 4H), 7.82 (d, 2H, J = 9.20 Hz), 7.90 (s, 1H), 8.08 (d,

2H, J = 8.85 Hz), 8.45 (d, 2H, J = 9.20 Hz) MS (ESI+) calculated for [M+H]+: 811.34 Da, Found

811.44 Da. See appendix A for the spectra.

3-azidopropan-1-ol (5): 3-azidopropan-1-ol (5) was prepared according to literature54. 28 mg

(0.17 mmol) of potassium iodide was added to 5 mL deionized water and stirred at room

temperature for 5 minutes. Then 1.44 grams (22.1 mmol) of sodium azide and 792 mg (8.37 mmol)

39

of 3-chloro-1-propanol were added. The mixture was refluxed (100oC) for 22 hours. The mixture

was then cooled to room temperature and extracted with diethyl ether (3x12.5 mL). The organic

layers were combined and washed with brine. The organic phase was dried with sodium sulfate

and the solvent was removed in vacuo to obtain the desired product as a clear oil (202 mg (1.99

mmol), yield 23.9%). 1HNMR (400 MHz DMSO-d6): 1.61 (t, 1H), 1.83 (p, 2H), 3.45 (t, 2H), 3.76

(q, 2H). See appendix A for the spectra.

3-azido-N-methylpropan-1-amine (6): 3-azido-N-methylpropan-1-amine (6) was prepared

according to literature54. To a sample of 190 mg (1.88 mmol) of 3-azidopropan-1-ol (5) in 7.5 mL

anhydrous dichloromethane on ice, 0.8 mL (5.5 mmol) triethylamine was added. Then, 0.25 mL

(3.23 mmol) of methanesulfonyl chloride was slowly added to the mixture and stirred for 5 minutes

at 0oC. The solution was stirred for an additional 20 minutes at room temperature and thereafter

the solvent was removed in vacuo. The obtained crude product was dissolved in 15 mL anhydrous

tetrahydrofuran (THF), 75 𝜇L (0.6 mmol) of triethylamine and 3.8 mL (43.8 mmol) of

methylamine (40% in water). The solution was refluxed for 20 hours at 65oC. The solution was

then distilled with a distillation bridge at 90oC at ambient pressure. The obtained distillate was

concentrated in vacuo to obtain the desired product as a faint yellow oil (80 mg (0.7 mmol), yield

37.3%). 1HNMR (400 MHz DMSO-d6): 1.72 (m, 2H), 2.38 (s, 3H), 2.62 (t, 2H), 3.32 (t, 2H). See

appendix A for the spectra.

RB-N3 (7): Under argon, 24 mg (0.02 mmol) of Rose Bengal was added to 1.5 mL anhydrous

DMF and stirred 0oC for 2 minutes. Then, 0.01 mL (0.07 mmol) of triethylamine and 10 mg (0.03

mmol) of HATU were added to the reaction mixture and stirred at 0oC for 5 minutes. Finally, 10

mg (0.03 mmol) of 3-azido-N-methylpropan-1-amine (6) was added to the red solution and the

40

mixture was stirred at 0oC for 2 hours. The mixture was then stirred overnight at 40oC. The solvent

was removed in vacuo and the obtained crude product was purified via flash chromatography

(ethyl acetate with 10% methanol) to obtain the desired product as a red powder (15 mg (0.014

mmol), yield 60.9%). MS (ESI-) calculated for [M-H]-: 1068.57 Da, Found 1068.79 Da. See

appendix A for the spectra.

P1 (8): A stock solution of copper (I) iodide was prepared by combining a solution (25 mg – 0.100

mmol in 1 mL DDI water) of copper (II) sulfate pentahydrate and sodium ascorbate (25 mg – 0.126

mmol in 1 mL DDI water). Then, 2.9 mg (0.004 mmol) of RB-N3 (7) was added to 160 𝜇L

anhydrous acetonitrile. Next, 0.5 mL of the copper (I) iodide stock solution was added to the

mixture and stirred at 40oC overnight. The solvent was removed in vacuo and the crude product

was subsequently purified via flash chromatography (ethyl acetate with 10% methanol to ethyl

acetate with 15% methanol and 2% acetic acid) to obtain the desired product as a purple powder

(6.00 mg (0.003 mmol), yield 89.1%). MS (ESI-) calculated for [M-H]-: 1879.67 Da, Found

1879.80 Da. See appendix A for the spectra.

CF3 Protected 4-Methylamine Benzyl Alcohol (9): CF3 Protected 4-Methylamine Benzyl

alcohol was prepared according to literature55. Under argon, 1 gram (7.35 mmol) of 4-methylamine

benzyl alcohol was added to a round bottom flask with 9 mL anhydrous methanol and 0.9 mL

41

(6.45 mmol) of triethylamine. Next, 1 mL (9.65 mmol) of ethyl trifluoroacetate was slowly added

to the round bottom flask and the reaction was stirred at room temperature for 1.5 hours. Then, the

solvent was removed in vacuo and the obtained crude product was purified via flash

chromatography (2:1 ethyl acetate to hexane) to obtain the desired product as a white powder (1.55

g, (6.65 mmol) yield 90 %). 1HNMR (400 MHz CDCl3): 1.67 (t, 1H), 4.54 (d, 2H, J = 6.21 Hz),

4.72 (d, 2H, J = 4.48 Hz), 6.52 (bs, 1H), 7.34 (d, 4H, J = 26.65 Hz). See appendix A for spectra.

CF3 Protected 4-Methylamine O6 Benzyl Guanine (10): Under argon, 356 mg (1.53 mmol) of

compound 9 was added to a round bottom flask with 7.5 mL anhydrous DMF and stirred at room

temperature for 2 minutes to obtain a yellow solution. Then, 694 mg (6 mmol) of potassium tert-

butoxide was added resulting in a dark red/maroon solution. Next, 205 mg (0.81 mmol) of guanine

salt (compound 1) was added, and the mixture was stirred at room temperature for 3 hours. The

solvent was removed in vacuo. The sample was then dry loaded on to a column and further purified

via flash chromatography (100% dichloromethane to 95% dichloromethane with 5% methanol) to

obtain the desired product as a yellow solid (161 mg (0.81 mmol), yield 54.7%).1HNMR (400

MHz DMSO-d6): 4.40 (d, 2H, J = 6.38 Hz), 5.46 (s, 2H), 6.28 (s, 2H), 7.31 (d, 2H, J = 7.58 Hz),

7.48 (d, 2H, J = 8.71 Hz), 7.80 (s, 1H), 10.00 (s, 1H), 12.40 (s, 1H). See appendix A for the

spectra.

4-Methylamine O6 Benzyl Guanine (11): Under argon, 80 mg (0.218 mmol) of compound 10

was added to a round bottom flask with 4 mL anhydrous methanol and stirred at 60oC until a clear

solution was obtained. Then, 2 mL (33.1 mmol) ammonia solution (32% in water) was added to

42

the solution and the mixture was stirred at 40oC for 3 hours. The reaction completion was

monitored with a TLC accompanied by ninhydrin staining (8:2, DCM/MeOH). The solvent was

removed in vacuo and the crude product was used without further purification.

Sarcosine Linked Pyro A (12): Under argon, 5.4 mg (0.010 mmol) of Pyropheophorbide A was

added to 0.5 mL anhydrous DMF and stirred at 0oC for 2 minutes. Then, 0.05 mL (0.355 mmol)

of triethylamine and 4.6 mg (0.012 mmol) of HATU were added to the reaction mixture and stirred

at 0oC for 5 minutes. Finally, 1 mg (0.011 mmol) of sarcosine was added to the green solution and

the mixture was stirred at 0oC for 2 hours. The mixture was then stirred overnight at 40oC. The

solvent was removed in vacuo and the obtained crude product was used without any further

purification. MS (ESI-) calculated for [M-H]-: 604.74, Found 604.56. MS (ESI+) calculated for

[M+H]+: 606.74 Da, Found 606.54 Da. See appendix A for the spectra.

P1NQ (13): Under argon, 5 mg (0.008 mmol) of compound 12 was added to 0.5 mL anhydrous

DMF and stirred at 0oC for 2 minutes. Then, 0.1 mL (0.355 mmol) of triethylamine and 3.7 mg

(0.01 mmol) of HATU were added to the reaction mixture and stirred at 0oC for 5 minutes. Finally,

43

2.5 mg (0.01 mmol) of compound 12 was added to the green solution and the mixture was stirred

at 0oC for 2 hours. The mixture was then stirred overnight at 40oC. The solvent was removed in

vacuo and the obtained crude product was purified via HPLC (H2O with 0.1% TFA and MeOH

with 0.1% TFA). MS (ESI+) calculated for [M+H]+: 858.41 Da, Found 858.73 Da. See appendix

A for the spectra.

44

Chapter 3 A Hypoxia activatable Photosensitizer

Nitroreductase (NTR) as a PDT target

One of the most well-known characteristics of cancer is rapid, uncontrolled cell division. To

achieve such aberrant growth, the tumor body needs an active supply of nutrients and oxygen.

However, such supply is not always available since tumor growth often exceeds the development

of surrounding vasculature, leading to pockets of low oxygen or hypoxia56,57. These hypoxic zones

lower the effectiveness of chemotherapy by inhibiting the creation of oxygen-based radicals that

are key in inducing cell death via DNA damage56,57. The absence of the appropriate vasculature in

such regions also prevents the adequate delivery of anticancer agents and hence lowers their

potency56,57. Furthermore, hypoxia promotes metastic behavior in cancer cells via loss of adhesion

molecules such as E-cadherin and as a result, decreases the efficacy of surgical treatment56–58.

Fortunately, the aberrant growth, characteristic of tumor bodies, and the resultant hypoxia are

unique to cancer cells, that is, they do not occur under physiological conditions in healthy cells57.

Therefore, the hypoxic microenvironment is an exclusive feature of cancer cells that may be

exploited to gain selective treatment. The lack of oxygen in the tumor body results in a much more

reducing environment compared to healthy cells due to the upregulation of several different types

of reductase enzymes56,57,59. One such family of reductases that are known to be upregulated in

hypoxia and therefore serve as an important cancer biomarker, is Nitroreductase (NTR)34,59,60.

NTR is a family of flavin enzymes, responsible for the reduction of nitro groups, especially

nitroarenes, to their amino counterparts34,59–61. Based on their mechanism of action, the NTR

family can be split into two different groups; type I and type II. The type I NTR (oxygen-insensitive

NTR), reduce the nitro group to the nitroso counterpart via a direct 2 electron transfer (Figure

3.1)60. The resultant nitroso compound is then further reduced to a hydroxylamine and finally the

amine counterparts with the aid of a 2-electron donor, reduced nicotinamide adenine dinucleotide

(NADH) (Figure 3.1)60. On the other hand, type II NTR (oxygen-sensitive NTR) covert the nitro

group to the nitroso counterpart via 2 separate single electron transfer steps, proceeding through

the formation of a nitro anion radical (Figure 3.1)60. Much like the type I NTR mechanism, the

nitroso compound can be further reduced to the hydroxylamine and then the amine with the use of

NADH (Figure 3.1)60. However, in the case of type II NTR this complete reduction is only possible

in a hypoxic environment, since the presence of oxygen leads to the quenching of the nitro anion

45

radical via an electron transfer, reverting the compound back to the nitro form and producing

superoxide anion radicals60.

Figure 3.1. Mechanism of action for the reduction of nitro groups by type I and type II Nitroreductase (NTR).

This image was obtained from [60].

This hypoxia driven conversion of an electron withdrawing group to an electron donor has been

exploited in the development of bio-reductive prodrugs57 and type II NTR activatable

fluorescencent chemosensors for cancer detection59. Unfortunately, the utility of this cancer

biomarker for development of an activatable PS probe is yet to be realized. One possible reason

for this is the misconception that photodynamic therapy (PDT) cannot occur in a hypoxic

environment. While it is true that oxygen is needed to produce the active cytotoxic agent, singlet

oxygen, PDT does not necessarily need ambient concentrations (20%) of oxygen. In fact, PDT has

been shown to work just as efficiently at about 5% oxygen, or under mild hypoxia relative to

normoxia (20%)62. This principle was demonstrated in recent work by the Urano group who

developed a mild hypoxia activatable PS probe based upon the reductive action of an enzyme

overexpressed in hypoxia, Azoreductase63. Based upon on their work, it was proposed that NTR

may also be potentially utilized in an analogous fashion to develop a mild hypoxia, NTR

activatable PS. In designing such a probe, it was found that a methylene blue (MB) based NTR

activatable probe, p-NBMB, had already been developed by the Jo group (Figure 3.2)34. The group

utilized a caging technique to trap the MB in a non-photoactive leuco state by modifying the imine

with an NTR removable scaffold, such that, the aromaticity of the chromophore was lost (Figure

3.2)34. Upon reduction of the nitro group at the para position of the benzyl carbamate scaffold to

46

the amine counterpart, a spontaneous 1,6-rearrangement was triggered, resulting in the elimination

of the cage scaffold and the liberation of the photoactive MB (Figure 3.2)34.

Figure 3.2. Mechanism of the NTR mediated activation of p-NBMB. NTR transfers a single electron to the nitro

group to yield a nitro anion radical. Under hypoxic conditions, NTR may transfer a second electron and with

subsequent aid from NAD(P)H, reduce the nitro anion radical to the amine counterpart. The amine scaffold

then undergoes a 1,6-rearrangement to self-eliminate and liberate the leuco methylene blue which is

spontaneously oxidized by the molecular oxygen to the photoactive MB variant. This image was obtained from

[34].

However, p-NBMB was presented as a fluorescent chemosensor for NTR detection in anaerobic

bacteria and not as an activatable PS34. Thus, we sought out to determine whether p-NBMB could

be used for PDT applications.

Results and discussion

p-NBMB, was synthesized as described by Jo and associates (Figure 3.3)34. The first step of the

synthesis involved the reduction of the photoactive methylene blue (MB) to the non-photoactive

leuco MB derivative using the strong reductant, sodium dithionite in a water-toluene mixture.

Upon reduction, the organic phase was combined with the enzyme trigger group to yield the

desired caged probe. For details on the synthesis, refer to the materials and methods section below.

47

Figure 3.3. The synthetic scheme for the synthesis of p-NBMB as described by Jo and associates34.

Once the probe was synthesized, a UV-Visible spectroscopy-based assay was performed to

examine enzyme induced activatability. As shown in Figure 3.4 (red curve), the UV-Visible

spectrum of p-NBMB in PBS with NADH at 37oC, lacked the absorbance peak at 667 nm

characteristic of the photoactive MB. However, upon addition of Nitroreductase (NTR), an

increase in absorbance at 667 nm was observed within 5 minutes, with a plateau occurring at 1

hour (Figure 3.4 – blue curve). Based upon this data, it appeared NTR was capable of activating

p-NBMB to the photoactive form (MB) as reported by Jo and associates34.

Figure 3.4. Activatability assay using UV-Visible spectroscopy. Leuco MB probe (p-NBMB) (1.7 𝛍𝐌) with 0.1

mM NADH, exhibited no absorbance at 667 nm in the absence of NTR (red curve). After an hour incubation

with NTR (280 n𝐌) an absorbance peak at 667 nm was observed (blue curve).

48

A UV-Visible kinetic assay was also performed to confirm the time taken for the complete

activation of p-NBMB. As shown in Figure 3.5, consistent with the first activatability assay (Figure

3.4), it took about 1 hour to achieve a final fold change of approximately 11-fold. A control assay

was also performed in the absence of the NTR (Figure 3.6) to ensure the activation was driven by

the enzyme. As shown in Figure 3.6, an increase in absorbance at 667 nm was found to occur in

the absence of the NTR as well. However, measurement of the rates of increase in absorbance at

667 nm showed that the increase in the absence of NTR (1.45X10-5 Au/sec) was 10-fold slower

than in the presence of NTR (1.86X10-4 Au/sec). As such, NTR was responsible for the removal

of the cage group resulting in the liberation of the photoactive MB.

Figure 3.5. Measurement of the change in absorbance at 667 nm as a function of time for p-NBMB (1.7 𝛍𝐌) in

PBS with 0.1 mM NADH and 280 n𝐌 NTR. A plateau was observed after approximately 1 hour of incubation

at 37oC.

49

Figure 3.6. Measurement of the change in absorbance at 667 nm as a function of time for p-NBMB (1.7 𝛍𝐌)

in PBS with 0.1 mM NADH (no NTR).

Once the liberation of the photoactive MB from p-NBMB by NTR was confirmed, an investigation

of the NTR induced generation of singlet oxygen by p-NBMB was performed via a fluorescence

assay based using the singlet oxygen sensor green (SOS-G)64. As shown in Figure 3.7, SOS-G

features a chlorinated fluorescein moiety covalently attached to a methyl anthracene (MA)

scaffold. In this form, the MA acts as a quencher for fluorescein64. However, when singlet oxygen

is generated, the MA scaffold is oxidized, forming an endoperoxide such that the quenching of the

fluorescein is diminished, resulting in an increase in fluoresence64. If singlet oxygen generation by

p-NBMB is NTR activatable, an increase in fluorescence emission from SOS-G (λem – 525 nm)

should only be observed upon irradiation of the p-NBMB sample in the presence of NTR. As

shown in Figure 3.8, irradiation of the p-NBMB containing SOS-G sample without NTR did not

induce an increase in fluorescence, indicating the inability of the caged MB to produce singlet

50

oxygen. Meanwhile, a 1.5-hour incubation of the sample with NTR, with subsequent irradiation

of the sample yielded a 1.5-fold increase in fluorescence indicative of singlet oxygen production

(Figure 3.9).

Figure 3.8. Fluorescence emission of SOS-G (1 μM) in a sample of 5 μM p-NBMB, 0.1 mM NADH in 50%

PBS/D2O mixture with 1% DMSO, without irradiation (red curve) and with 5 minutes irradiation (blue curve)

at 625 nm (2.6 mW).

Figure 3.7. A schematic of the mechanism of activation for the singlet oxygen sensor green (SOS-G). The

sensor is weakly fluorescent due to electronic quenching of the fluorescein fluorophore by methyl

anthracene (MA). The quenching is diminished upon oxidation of the MA scaffold by singlet oxygen,

leading to an increase in fluorescence. This Figure was obtained from [64].

550 600 6500

20

40

60

80

Singlet Oxygen Assay

Wavelength

Flu

ore

sen

ce I

nte

nsi

ty

NTR - 90 Mins - No Irr

NTR - 90 Mins - 5 Mins Irr

51

Figure 3.9. Fluorescence emission of SOS-G (1 μM) in a sample of 5 μM p-NBMB, 0.1 mM NADH in 50%

PBS/D2O mixture with 1% DMSO after 90 minutes incubation with 0.28 µM NTR, without irradiation (red

curve) and with 5 minutes irradiation (blue curve) at 625 nm (2.6 mW).

To validate the results of the SOS-G assay, a second fluorescence assay based upon the reactive

oxygen species (ROS) sensor, 2′,7′-Dichlorodihydrofluorescein diacetate (DCFH-DA) was

performed65. As shown in Figure 3.10, DCFH-DA is a reduced dichlorofluorescein chromophore

with acetate protected xanthene hydroxyls to endow cell permeability65. For the desired in vitro

assay, the acetate groups must first be cleaved using a solution of 0.01 N sodium hydroxide

(NaOH), to liberate the active reduced non-fluorescent ROS sensor, DCFH (Reduced

dichlorofluorescein) (Figure 3.10)65. If ROS species are produced by the molecule of interest, the

reduced non-fluorescent chromophore (DCFH) is then oxidized to its fluorescent analogue, DCF

(Figure 3.10)65. If singlet oxygen generation by p-NBMB is NTR activatable, an increase in

fluorescence emission from DCFH (λem – 522 nm) should only be observed upon irradiation of the

p-NBMB sample post incubation with NTR. As demonstrated in Figure 3.11, irradiation of the

DCFH sample with only p-NBMB produced a 3.2-fold increase in fluorescence. This background

increase in fluorescence was attributed to spontaneous oxidation of DCFH to DCF by the

environment. On the other hand, irradiation post 1.5-hour incubation of the sample with NTR was

found to induce a 6.5-fold increase in fluorescence (Figure 3.12). This fluorescence increase

52

indicated the successful generation of singlet oxygen species (a type of ROS), consistent with the

SOS-G assay. Therefore, it was concluded that p-NBMB exhibits NTR activatable behavior.

Figure 3.10. A schematic of the mechanism of activation of the ROS sensor, DCFH-DA. For in vitro testing, the

diacetate groups must first be cleaved from the scaffold to liberate the active non-fluorescent sensor, DCFH. In

the presence of ROS species, DCFH is oxidized to its fluorescent analogue DCF. This Figure was obtained from

[65].

Figure 3.11. Fluorescence emission of DCFH (1 μM) in a sample of 5 μM p-NBMB, 0.1 mM NADH in 50%

PBS/D2O mixture with 1% DMSO, without irradiation (brown curve) and with 5 minutes irradiation (green

curve) at 625 nm (2.6 mW).

550 600 6500

20

40

60

80

ROS Assay

Wavelength

Flu

ore

sen

ce I

nte

nsi

ty

DCFH - No Irr

DCFH - 5 Mins Irr

53

Figure 3.12. Fluorescence emission of DCFH (1 μM) in a sample of 5 μM p-NBMB, 0.1 mM NADH in 50%

PBS/D2O mixture with 1% DMSO after 90 minutes incubation with 0.28 µM NTR, without irradiation (red

curve) and with 5 minutes irradiation (blue curve) at 625 nm (2.6 mW).

Future directions & experiments

As demonstrated in the in vitro analysis above, p-NBMB is an NTR activatable MB probe with

enzyme triggered singlet oxygen production. Although this is a promising foundation, it should be

noted that the in vitro analysis is performed with an oxygen insensitive, type I NTR. Therefore,

further in cellulo analysis needs to be performed to validate the utility of p-NBMB as a PDT probe

in a hypoxic setting.

To begin, the cellular permeability and dark toxicity of p-NBMB and the parent MB dye should

be investigated in several different cell lines known to overexpress NTR under hypoxia such as

A549 and MCF-7. Once the dark toxicity threshold has been determined, the appropriate

concentrations of p-NBMB should be incubated with cells under normoxia and various extents of

hypoxia with subsequent irradiation to measure difference in phototoxicity. Since the employed

cell lines overexpress NTR under hypoxia, p-NBMB should only be phototoxic under such

conditions. If such results are obtained, the efficacy of p-NBMB should be further examined in a

mouse models.

550 600 6500

200

400

600

800

1000

ROS Assay

Wavelength

Flu

ore

sen

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nsi

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DCFH - NTR - No Irr

DCFH - NTR - 5 Mins Irr

54

Materials & methods

General Methods

All reactions were conducted in oven-dried glassware under atmospheric conditions, without the

use of inert gas protection. All reagents for which the synthesis is not provided were purchased

from Sigma Aldrich or Alfa Aesar and used as purchased without further purification. Final probe

solutions were prepared using high grade DMSO from Thermo Fisher Scientific. PBS pH 7.4 (1X)

buffer was purchased from Thermo Fisher Scientific. Fluorescent spectra were obtained using a

Shimadzu RF-6000 Spectro fluorophotometer, while UV-Visible spectra were obtained using a

Shimadzu UV-1800. Irradiation of the sample was conducted using the 625 nm mounted LED by

Thorlabs. The concentration of p-NBMB was estimated using the extinction coefficient of

methylene blue at 665 nm (91000 M-1cm-1) as reported in literature13.

UV-Visible NTR Activatability Assay

Using a 60 𝜇L quartz cuvette, the UV-Vis spectrophotometer was blanked with PBS pH 7.4. Then,

0.6 μL of a 172 μM p-NBMB stock solution (in DMSO) and 6 μL of a 1 mM NADH stock solution

(in DDI H2O) were added to 52.8 μL PBS buffer to obtain a UV-Vis spectrum of p-NBMB (final

concentration 1.72 μM). After 5 minutes of incubation a second UV-Vis spectrum was obtained

to ensure no change due to stability of the caged probe. Then, 0.6 μL of a 28 μM NTR stock

solution (final concentration 280 nM) was added and UV-Vis spectra were collected every 5

minutes until no further change in the spectrum was observed (60 minutes).

For the kinetic experiment, the procedure was repeated as mentioned above but by monitoring the

change at 667 nm as a function of time. The rates of change were calculated by measuring the

initial rates.

Singlet-Oxygen Production

Without NTR: 0.6 𝜇L of SOS-G (100 𝜇M in methanol) (final concentration 1 𝜇M), 0.6 μL of a

500 μM p-NBMB stock solution (in DMSO) and 6 μL of a 1mM NADH stock solution (in DDI

H2O) were added to a 60 𝜇L quartz cuvette containing 30 𝜇L D2O and 22.8 𝜇L pH 7.4 PBS buffer

at 37oC. A fluorescent spectrum was obtained (λ𝑒𝑥 = 495 nm, λ𝑒𝑚 = 510 to 650 nm) (3nm

excitation and emission slit width). The sample was irradiated at 625 nm (2.6 mW) for 5 minutes.

55

Another fluorescent spectrum was obtained to measure any changes due to irradiation of the

sample in the absence of NTR.

With NTR: 0.6 𝜇L of SOS-G (100 𝜇M in methanol) (final concentration 1 𝜇M), 0.6 μL of a 500

μM p-NBMB stock solution (in DMSO), 6 μL of a 1 mM NADH stock solution (in DDI H2O) and

0.6 μL of a 28 μM NTR stock solution (final concentration 280 nM) were added to a 60 𝜇L quartz

cuvette containing 30 𝜇L D2O and 22.2 𝜇L pH 7.4 PBS buffer. The mixture was incubated at 37oC

for 90 minutes. A fluorescent spectrum was obtained (λ𝑒𝑥 = 495 nm, λ𝑒𝑚 = 510 to 650 nm) (3nm

excitation and emission slit width). The sample was then irradiated at 625 nm (2.6 mW) for 5

minutes and a second fluorescent spectrum was obtained to measure any changes due to irradiation

of the sample in the presence of NTR.

ROS production

Without NTR: A 20 𝜇M stock solution of the active ROS sensor (DCFH-DH) was prepared by

diluting a 100 𝜇M stock solution of the diacetate derivative of the ROS sensor in 0.01 N sodium

hydroxide. Next, 3 𝜇L of the active ROS sensor solution, 0.6 μL of a 500 μM p-NBMB stock

solution (in DMSO) and 6 μL of a 1 mM NADH stock solution (in DDI H2O) were added to a 60

𝜇L quartz cuvette containing 20.4 𝜇L pH 7.4 PBS buffer and 30 𝜇L D2O at 37oC. A fluorescent

spectrum was obtained (λ𝑒𝑥 = 495 nm, λ𝑒𝑚 = 510 to 650 nm) (3nm excitation and emission slit

width). The sample was irradiated at 625 nm (2.6 mW) for 5 minutes. Another fluorescent

spectrum was obtained to measure any changes due to irradiation of the sample in the absence of

NTR.

With NTR: A 20 𝜇M stock solution of the active ROS sensor (DCFH-DH) was prepared by

diluting a 100 𝜇M stock solution of the diacetate derivative of the ROS sensor in 0.01 N sodium

hydroxide. Next, 3 𝜇L of the active ROS sensor solution, 0.6 μL of a 500 μM p-NBMB stock

solution (in DMSO), 6 μL of a 1 mM NADH stock solution (in DDI H2O) and 0.6 μL of a 28 μM

NTR stock solution (final concentration 280 nM) were added to a 60 𝜇L quartz cuvette containing

19.8 𝜇L pH 7.4 PBS buffer and 30 𝜇L D2O. The mixture was incubated at 37oC for 90 minutes. A

fluorescent spectrum was obtained (λ𝑒𝑥 = 495 nm, λ𝑒𝑚 = 510 to 650 nm) (3nm excitation and

emission slit width). The sample was irradiated at 625 nm (2.6 mW) for 5 minutes. Another

56

fluorescent spectrum was obtained to measure any changes due to irradiation of the sample in the

presence of NTR

Synthesis (Refer to Figure 3.3 for full scheme)

p-NBMB (1): p-NBMB was prepared according to literature34. Under argon, 319 mg (1.00 mmol)

of Methylene blue (MB) was added to 10 mL of deionized water and 40 mL of toluene. Then, 294

mg (3.50 mmol) of sodium bicarbonate and 496 mg (2.85 mmol) of sodium dithionite were added

to the mixture and stirred at 60oC for 30 minutes behind a bomb shield. After the 30 minutes, the

leuco MB containing toluene phase was slowly transferred to a new vessel with 215 mg (1.00

mmol) of 4-Nitrobenzyl chloroformate dissolved in 40 mL toluene, maintained in an ice bath. The

mixture was allowed to stir overnight. The organic solution was then washed 5X with 30 mL

deionized water and dried with sodium sulphate. The toluene was then removed in vacuo and the

product was precipitated with cold methanol to obtain p-NBMB as orange crystals (2.5 mg (0.005

mmol), yield 0.5%). MS (ESI+) calculated for [M+H]+: 465.54 Da, Found 465.30 Da. See

appendix A for the spectra.

57

Chapter 4 3AIC: A Novel Coumarin Based Photosensitizer

Why does the world need a coumarin photosensitizer (PS)?

The term photodynamic was first used in 1903 by Herman Von

Tappeiner and A. Jesionek, who topically applied eosin (Figure 4.1)

in combination with white light to combat skin cancer18. Since then,

the field of photodynamic therapy (PDT) has emerged into an

extremely powerful tool to combat cancer, with several

photosensitizer drugs (PSs) gaining clinical approval in various

countries13,18,20. Unfortunately, as highlighted in the previous

chapter, the clinical utility of PDT for cancer remains limited. One

of the key hinderances in widespread adoption of PDT is the absence of structural features on

clinically approved PSs, which allow for differentiation between healthy and cancer cells13. As a

result, current clinically approved PSs induce phototoxicity in healthy cells adjacent to the cancer

cells during irradiation. In addition, the tetrapyrrole family of PSs, which makes up most clinically

approved PSs, suffer from poor water solubility and poor clearance leading to prolonged skin

sensitivity19,20,24. Furthermore, although commercially available, many porphyrins with PDT

properties (such as Pyropheophorbide-a) are extremely expensive and synthetically challenging to

prepare. On the other hand, non-tetrapyrrole based PSs such as methylene blue and Rose Bengal

suffer from excessive dark toxicity and poor photostability, respectively19,25. These limitations of

pre-existing PSs act to bottleneck the development of targeting strategies via structural

improvements, which may endow these molecules with greater selectivity for cancer cells over

healthy cells and thereby reduce toxicity. As such, the development of novel PSs with good

photostability and water solubility, that can be prepared in a facile manner with minimal

purification are highly warranted. Such PSs would allow for the development of targeting

strategies on a proof of principle basis, which may then be further extrapolated to other pre-existing

PSs. In this chapter, the facile development and subsequent photophysical characterization of a

novel PS based upon an iodinated 3-aminocoumarin scaffold is discussed. The coumarin

chromophore is ideal for the development of a PS, as the scaffold is known to be bioactive with an

abundance of literature on synthesis. In fact, one of oldest family of PSs, psoralens, is based upon

a furanocoumarin structure (Figure 4.2)66. Although, the psoralens are capable of singlet oxygen

Figure 4.1. Chemical Structure

of Eosin.

58

production, their quantum yield of singlet oxygen is low (between

0.2-0.4)66. This limitation has indorsed the development of other

coumarin based PSs which have greater singlet oxygen quantum

yields66–68. Unfortunately, all these PSs require lengthy synthesis

with purification at each step. In addition, these coumarin based PSs

lack any functional handles (such as amine or carboxylic acid) which may be further exploited to

introduce targeting moieties for better selectivity. To improve upon such limitations, the

development of a novel coumarin based PS, 3-amino-6,8-diiodocoumarin (“3AIC”) was proposed.

Built upon a simple 3-aminocoumarin (3AC) structure, 3AIC features iodine atoms at the 6 and 8

positions of the scaffold, promoting intersystem crossing and therefore inducing singlet oxygen

production. In addition, the inclusion of an amine handle allows for future structural modifications

of the PS with targeting moieties to aid in the development of cancer targeted PSs.

Results and discussion

To begin, 3AIC was synthesized utilizing a combination of literature based upon the synthesis of

3AC (Figure 4.3)69–71. To minimize purification steps, synthetic protocols utilizing simple product

precipitation were found69–71. Then, to introduce iodine atoms at the desired 6 and 8 positions, the

synthetic pathway was modified to include the commercially available and inexpensive 3,5-

diiodosalicyladehyde instead of the non-iodinated analogue, salicylaldehyde. It is worth noting,

the synthetic pathway can be further shortened to two easy steps, if N-acetylglycine ($13.80

CAD)72 is simply purchased. For details on the synthesis, refer to the materials and methods section

below.

Once synthesized, a UV-Visible spectrum of 3AIC was obtained and compared to that of the

commercially available non-iodinated variant, 3AC. As shown in Figure 4.4, 3AIC was found to

have a similar shape to 3AC but the inclusion of the iodines appeared to induce a slight

bathochromic shift. Note, the samples of 3AC and 3AIC were prepared to have the same

absorbance at 365 nm (Figure 4.4), which will be the wavelength used for photoexcitation in

subsequent singlet oxygen and ROS generation assays.

Figure 4.2. Chemical Structure

of Psoralen.

59

Figure 4.3. Synthetic scheme for the synthesis of 3AIC and associated intermediates.

Figure 4.4. The UV-Visible spectrum of 3-aminocoumarin (3AC) in the blue curve and 3-amino-6,8-diiodocoumarin (3AIC)

in the black curve in PBS with 1% DMSO. Note, the concentrations of both samples 3AC (109 μM) and 3AIC (8.25 μM)

were used such that the both compounds had the same absorbance at 365 nm (the wavelength used for photoexcitation).

To measure the singlet oxygen production, a fluorescence assay based upon the singlet oxygen

sensor green (SOS-G) was performed64,73. For details on the SOS-G assay refer to chapter 3. In the

case of singlet oxygen generation by 3AIC, if SOS-G and 3AIC are incubated together and the

sample is irradiated with a 365 nm LED, then if singlet oxygen is produced, one should observe

an increase in fluorescence at the emission wavelength of fluorescein (λ𝑒𝑚 = 525 nm). As shown

in Figure 4.5A, irradiation of SOS-G (1 μM) at 365 nm for 5 minutes, did not induce an increase

in fluorescence emission at 525 nm. However, when 3AIC (8.25 μM) was present in the solution

60

mixture, irradiation for 5 minutes at 365 nm, resulted in an increase in fluorescent emission at 525

nm, as would be expected if singlet oxygen was produced (Figure 4.5B).

A

B

To confirm the facilitation of intersystem crossing by the presence of iodine atoms, a further SOS-

G singlet oxygen assay was performed with the non-iodinated 3AC scaffold. As shown in Figure

4.6, no increase in fluorescence from SOS-G (1 μM) was observed upon irradiation either in the

absence (Figure 4.6A) or presence (Figure 4.6B) of the 3AC (109 μM), indicating that the non-

iodinated scaffold cannot produce singlet oxygen. In other words, the intersystem crossing

promoted by the iodines at the 6 and 8 positions of 3AIC is imperative in its ability to act as a PS.

Figure 4.5. A) Fluorescence emission of SOS-G (1 μM) in the absence of 3AIC, without irradiation (red curve) and with

irradiation (1.1 mW) (blue curve) at 365 nm in 50% PBS/D2O mixture with 1% DMSO. B) Fluorescence emission of SOS-

G (1 μM) in the presence of 3AIC (8.25 μM), without irradiation (red curve) and with irradiation (1.1 mW) (blue curve) at

365 nm in 50% PBS/D2O mixture with 1% DMSO. Refer to the materials and methods section for details.

61

A

B

Next, the results of the SOS-G singlet oxygen assay were further validated via investigation of the

reactive oxygen species (ROS) production by 3AIC using a general ROS sensor, 2′,7′-

Dichlorodihydrofluorescein diacetate (DCFH-DA)65. For details on the DCFH-DA ROS assay,

refer to chapter 3. If ROS species are produced by 3AIC, one should observe an increase in

fluorescent emission at 522 nm. As shown in Figure 4.7B, 5-minute irradiation of a sample

containing both DCFH (1 μM) and 3AIC (8.25 μM), resulted in an approximately 43-fold increase

in fluorescence at 522 nm, relative to the emission of a sample without irradiation. The ROS assay

supported the data observed in the SOS-G assay, that is, 3AIC is a proficient PS capable of

producing singlet oxygen and potentially other ROS species as well. It is worth noting, that the

Figure 4.6. A) Fluorescence emission of SOS-G (1 μM) in the absence of 3AC, without irradiation (red curve) and with

irradiation (1.1 mW) (green curve) at 365 nm in 50% PBS/D2O mixture with 1% DMSO. B) Fluorescence emission of SOS-

G (1 μM) in the presence of 3AC (109 μM), without irradiation (brown curve) and with irradiation (1.1 mW) (blue curve)

at 365 nm in 50% PBS/D2O mixture with 1% DMSO. Refer to the materials and methods section for details.

62

oxidation required for the activation of DCFH, can occur simply due to spontaneous oxidation by

the atmospheric environment leading to a small background increase in fluorescence (about 3 to

4-fold) as shown in Figure 4.7A. The assay was repeated with the non-iodinated variant 3AC

(Figure 4.8), to confirm that the iodines are responsible for the PS properties of 3AIC. As shown

in Figure 4.8, the increase (~3 fold) observed in the fluorescence emission upon irradiation of the

sample containing 3AC (109 μM) and DCFH (1 μM), matched that of the background increase

upon the irradiation of DCFH (1 μM) in the absence of 3AC, indicating that 3AC cannot generate

ROS species. Therefore, consistent with the SOS-G data, 3AC does not have any PS properties.

A

B

550 600 650

-50

0

50

100

150

200

250

ROS Assay

Wavelength (nm)

Flu

ore

sen

ce I

nte

nsi

ty

No 3-AIC No Irr

No 3-AIC 5 Mins Irr

550 600 6500

2000

4000

6000

ROS Assay

Wavelength (nm)Flu

orese

nce I

nte

nsi

ty

3-AIC No Irr

3-AIC 5Mins Irr

Figure 4.7. A) Fluorescence emission of DCFH (1 μM) in the absence of 3AIC, without irradiation (blue curve) and with

irradiation (1.1 mW) (red curve) at 365 nm in 50% PBS/D2O mixture with 1% DMSO. Note, the background increase (3-

4 fold) appears to be due to spontaneous oxidation of the sensor by the atmospheric oxygen. B) Fluorescence emission of

DCFH (1 μM) in the presence of 3AIC (8.25 μM), without irradiation (blue curve) and with irradiation (1.1 mW) (red

curve) at 365 nm in 50% PBS/D2O mixture with 1% DMSO. Refer to the materials and methods section for details.

63

A

B

Since some iodinated PSs such as Rose Bengal are known to undergo photoinduced deiodination,

resulting in loss of PS properties25, the photostability of the 3AIC was investigated. As shown in

Figure 4.9, 3AIC (20 𝜇𝑀) was shown to undergo minimal change in absorbance upon intense

irradiation at 365 nm (1.1 mW) for up to 30 minutes. Thus, 3AIC is a photostable PS. In addition,

because these assays were performed in PBS buffer, it is clear 3AIC also exhibits water solubility.

550 600 6500

50

100

150

ROS Assay

Wavelength (nm)F

luorese

nce I

nte

nsi

ty No 3AC No Irr

No 3AC 5 Mins Irr

550 600 6500

100

200

300

400

ROS Assay

Wavelength (nm)

Flu

orese

nce I

nte

nsi

ty 3AC No Irr

3AC 5 Mins Irr

Figure 4.8. A) Fluorescence emission of DCFH (1 μM) in the absence of 3AC, without irradiation (blue curve) and with

irradiation (1.1 mW) (red curve) at 365 nm in 50% PBS/D2O mixture with 1% DMSO. Note, the background increase (3-4

fold) appears to be due to spontaneous oxidation of the sensor by the atmospheric oxygen. B) Fluorescence emission of

DCFH (1 μM) in the presence of 3AC (109 μM), without irradiation (blue curve) and with irradiation (1.1 mW) (red curve)

at 365 nm in 50% PBS/D2O mixture with 1% DMSO. Refer to the materials and methods section for details.

64

Future directions & experiments

Although the photostability and in vitro competence of 3AIC as a PS have been demonstrated, a

few more important experiments are yet to be conducted. To begin, concentrations of the 3AIC

have been estimated using the literature extinction coefficient value of the non-iodinated variant

3AC at 324 nm (13803 M-1cm-1) in methanol74. While structurally similar, the two compounds are

not identical, and hence the extinction coefficient of 3AIC should be experimentally determined

to ensure that the concentrations utilized in the above-mentioned experiments are accurately

described. Another in vitro experiment that remains to be completed, is the determination of the

quantum yield of singlet oxygen (Φ∆) of 3AIC and how that compares to other PSs. It is expected

that the efficient intersystem crossing brought on by the presence of the iodine atoms coupled with

the rigid structure will grant 3AIC a superior Φ∆ value in comparison to the psoralens which rely

solely on structural rigidity66.

Once these in vitro experiments have been conducted, the next task is to apply 3AIC to multiple

cancer cell lines and investigate the cell permeability, dark toxicity, and phototoxicity. To detect

Figure 4.9. 20 μM 3AIC in PBS (1% DMSO) irradiated at 365 nm (1.1 mW) for various time points.

65

the in cellulo singlet oxygen production, 3AIC must be applied in combination with DCFH-DA,

which upon singlet oxygen production will emit green fluorescence. If the cellular efficacy of

3AIC is deemed satisfactory, further structural modifications should be performed to introduce

cancer targeting moieties to endow greater selectivity for cancer cells vs. healthy cells.

Materials & methods

General Methods

All reactions were conducted in oven-dried glassware under atmospheric conditions, without the

use of inert gas protection. All reagents for which the synthesis is not provided were purchased

from Sigma Aldrich or Alfa Aesar and used as purchased without further purification. NMR

solvents were purchased from Cambridge isotope laboratories and used without further

purification. Final probe solutions were prepared using high grade DMSO from Thermo Fisher

Scientific. PBS pH 7.4 (1X) buffer was purchased from Thermo Fisher Scientific. NMR data was

collected using a Bruker Avance III 400 MHz spectrometer, with chemical shifts reported in ppm

relative to the residual solvent resonance peaks. Fluorescent spectra were obtained using a

Shimadzu RF-6000 Spectro fluorophotometer while UV-Vis spectra were obtained using a

Shimadzu UV-1800. Irradiation of the sample was conducted using 365 nm Mounted LED by

Thorlabs. The concentrations of 3AIC and 3AC stock samples were estimated using the extinction

coefficient of 3-aminocoumarin at 324 nm (13803 M-1cm-1) in methanol as reported in literature74.

UV-Vis Spectroscopy

Using a 60 𝜇L quartz cuvette, the UV-Vis spectrophotometer was blanked with pH 7.4 PBS. Then,

0.6 𝜇L of a 0.825 mM 3AIC stock solution (in DMSO) was added to 59.4 𝜇L PBS buffer to obtain

a UV-Vis spectrum of 3AIC (final concentration 8.25 𝜇M). The procedure was then repeated with

a stock solution of 3AC.

Singlet-Oxygen production

0.6 𝜇L of SOS-G (100 𝜇M in methanol) (final concentration 1 𝜇M) was added to a 60 𝜇L quartz

cuvette containing 30 𝜇L D2O and 29.1 𝜇L PBS pH 7.4 buffer. A fluorescent spectrum was

obtained (λ𝑒𝑥 = 495 nm, λ𝑒𝑚 = 510 to 650 nm) (3nm excitation and emission slit width). As a

control, the sample was irradiated at 365 nm (1.1 mW) for 5 minutes. Another fluorescent spectrum

66

was obtained to measure any changes due to irradiation of the singlet oxygen sensor. Next, 0.3 𝜇L

of 3AIC (0.825 mM in DMSO) (final concentration 4.13 𝜇M) was added to the cuvette. A

fluorescent spectrum was obtained after which, the sample was irradiated at 365 nm (1.1 mW) for

5 minutes. A final spectrum was obtained to investigate any changes due to the production of

singlet oxygen by 3AIC. The procedure was repeated for 3AC.

ROS production

A 20 𝜇M stock solution of the active ROS sensor (DCFH-DH) was prepared by diluting a 100 𝜇M

stock solution of the diacetate derivative of the ROS sensor in 0.01 N sodium hydroxide. Next, 3

𝜇L of the active ROS sensor solution was added to a 60 𝜇L quartz cuvette containing 25 𝜇L PBS

buffer pH 7.4 and 30 𝜇L D2O. A fluorescent spectrum of the sensor was obtained (λ𝑒𝑥 = 495 nm,

λ𝑒𝑚 = 510 to 650 nm) (3nm excitation and emission slit width). The sample was then irradiated at

365 nm (1.1 mW) for 5 minutes and another fluorescent spectrum was measured. Next, 2 𝜇L of a

3AIC stock solution (150 𝜇M in DMSO) (final concentration 5 𝜇M) was added to the cuvette and

the sample was irradiated at 365 nm (1.1 mW) for 5 minutes. A final fluorescent spectrum was

obtained to investigate any changes due to ROS production by 3AIC. The procedure was repeated

for 3AC.

Photostability

A 20 𝜇𝑀 solution of 3AIC in PBS (1% DMSO) was prepared in a 60 𝜇𝐿 quartz cuvette. The

sample was irradiated at 365 nm (1.1 mW) at 5-minute time intervals for up to 30 minutes and the

UV-Vis spectrum was obtained at each time point. The procedure was repeated 3 times and the

average absorbance values at 365 nm were plotted as a function of time.

Synthesis (Refer to Figure 4.3 for full scheme)

N-Acetylglycine (1): N-acetylglycine was synthesized according to literature69. 2.2 grams (29

mmol) of glycine in 10 mL of DI water were added to a round bottom flask. The mixture was

67

stirred at room temperature until the glycine was fully dissolved. Then, 5 mL (52 mmol) of acetic

anhydride was added to the mixture. The mixture was further stirred at room temperature for 30

minutes resulting in the round bottom becoming hot and the N-acetylglycine precipitating out. The

round bottom was then placed in the fridge (4 oC) overnight to complete precipitation. The

precipitate was isolated via vacuum filtration and washed with cold water to obtain the desired

product as a white solid (1.19 g (10.2 mmol), yield 35%). 1HNMR (400 MHz DMSO-d6): 1.84 (s,

3H), 3.72 (d, 2H, J = 6Hz), 8.16 (s, 1H), 12.48 (s, 1H). See Appendix 1 for spectra.

3AIC-Acetate (2): 3AIC-acetate was synthesized according to literature70, except 3,5-

diiodosalicyladehyde was used instead of salicylaldehyde. 870 mg (10.6 mmol) of sodium acetate

was added to a round bottom flask with 1.5 mL (15.9 mmol) acetic anhydride. The mixture was

stirred until the sodium acetate was fully dissolved. Then, 307 mg (2.62 mmol) N-Acetylglycine

was added to the round bottom followed by addition of 1 g (2.67 mmol) of 3,5-

diiodosalicyladehyde. The mixture was refluxed at 120oC for 3 hours. During this time, the

solution slowly turned brown and viscous. The mixture was allowed to cool to room temperature

to obtain a brown solid. Cold water was added to the precipitate and a spatula was used to break

up the solid. The slurry was then vacuum filtered and washed with cold water (2X). The precipitate

was then further washed with cold ethyl acetate to obtain the desired compound as an off white

solid (212 mg (0.466 mmol), yield 17.8%). 1HNMR (400 MHz DMSO-d6): 2.17 (s, 3H), 8.11 (d,

1H, J = 2.02 Hz), 8.19 (d, 1H, J = 2.02 Hz), 8.49 (s, 1H), 9.89 (s, 1H). See Appendix 1 for spectra.

3AIC (3): The synthesis of 3AIC was adapted from literature71. Briefly, an oil bath was preheated

to 120oC and 10 mL of a 70% H2SO4 solution (prepared by adding 5.8 mL of concentrated H2SO4

to 4.2 mL of DI water on ice) was added. Next, 35 mg (0.08 mmol) of 3AIC-acetate (compound

68

2) was added. The cloudy mixture was refluxed with continuous stirring until the solution turned

clear yellow (after approximately 1.5 hours). The solution was then allowed to come to room

temperature and 20.0 mL of ice cold water was added resulting in the precipitation of a white solid.

Next, about 25.0 mL of a cold 2N sodium hydroxide solution was added to the round bottom

resulting in further precipitation of a white solid. The precipitate was vacuum filtered, washed with

cold DI water and then dried under vacuum with gentle heating with a heat gun. The desired

product was isolated as an off white solid (22.0 mg (0.053 mmol), yield 69%). 1HNMR (400 MHz

CDCl3): 4.42 (s, 2H), 6.46 (s, 1H), 7.57 (s, 1H), 7.98 (s, 1H). See Appendix 1 for spectra.

69

Concluding Remarks

Photodynamic therapy (PDT) is a clinically approved cancer treatment which relies upon the

combined action of a photosensitizer drug (PS), light, and oxygen, to generate cytotoxic reactive

oxygen species to kill cells. The approach provides is an alternative to traditional cancer treatments

such as surgery and chemotherapy. Unfortunately, the clinical utility of this treatment remains

limited, partially due to the unselective nature of the PS drugs which cannot distinguish between

cancer and healthy cells, and thereby induce phototoxicity in healthy cells alongside cancer cells.

Improvement in the selective behavior of PSs is therefore imperative in increasing the clinical

utility of PDT. Pro-drug variants of pre-existing PSs that are activated by enzymes overexpressed

in cancer cells offer a potent solution. In this regard, the development and examination of two such

activatable PSs, Probe 1 and p-NBMB (Figure 5.1), targeted by the cancer biomarkers, O6-

methylguanine DNA methyltransferase (MGMT) and Nitroreductase (NTR), respectively, were

discussed in this body of work.

MGMT is a DNA repair enzyme that repairs alkylations at the O6-position of guanine by

transferring the alkyl group to a cysteine residue at its active site37,38. Several pseudosubstrates

have been developed which mimic alkylated guanine and thereby inhibit the enzyme. For

developing an MGMT activatable PS probe, one such inhibitor, O6-benzylguanine (O6-BG), was

modified with a commercially available PS, Rose Bengal, and the appropriate FRET quencher,

Figure 5.1. Structure of the examined enzyme activatable PS probes.

70

black hole quencher 2 (BHQ2), to generate a FRET quenched PS probe. The transfer of the PS

containing benzyl group at the O6 position of guanine to the enzyme active site should separate the

PS from the quencher, and thereby activate the PS. Unfortunately, such transfer was not observed.

To identify the source of this failure, the probe was dissected such that the quencher portion was

eliminated. In addition, the PS was switched from Rose Bengal to Pyropheophorbide-a due to

photostability issues associated with Rose Bengal25. The resultant always on probe, P1NQ (Figure

5.2), was found to successfully transfer the PS onto MGMT, albeit with poor efficiency (see

chapter 2). Nonetheless, P1NQ serves as a good starting point for the subsequent development of

functional and efficient FRET-based MGMT activatable PS probes. That being said, there are

some considerations with the FRET-based approach. The final probe with both the PS and the

quencher is going to be bulky and hydrophobic and as such, there may be issues in solubility and

cellular permeability. If the probe has too great of a hydrophobic character, it may be trapped

within the membrane and as such, would not be activated due to the lack of appropriate cellular

localization. Fortunately, such issues may be mitigated by introducing peg-based linkers between

the O6-BG and the PS and quencher to endow greater hydrophilic character. Alternatively, the PeT

quenching abilities of guanine may be also utilized with the appropriate PS dyes to develop a PeT-

based MGMT activatable PS probe, which does not require an accessary quencher and therefore

abolishes the introduction of excessive bulk and hydrophobicity.

Figure 5.2. The chemical structure of probe 1 no quencher (P1NQ).

The second target cancer biomarker, often overexpressed in hypoxia, exploited for activation of a

PS probe was NTR. It is responsible for the conversion of nitro groups (especially nitroarenes) to

their amine counterparts with the assistance of the 2-electron donor molecule, NADH59. Based

upon the action of the enzyme, a photoinactive derivative of methylene blue, called p-NBMB, with

a NTR specific 4-nitrobenzyl carbamate cage, was developed by the Jo group34. The probe was

used as a fluorescent sensor for NTR in anaerobic bacteria with no applications in the realm of

71

PDT34. Consequently, the probe was synthesized and subsequently investigated for its ability to

act as a NTR activatable PS probe. In vitro analysis of p-NBMB with NTR showed that the probe

can generate singlet oxygen only upon action of NTR (see chapter 3). However, in cellulo

evaluation of p-NBMB under various extents of hypoxia should be conducted to measure the

activatability and selectivity of the probe. In addition, the concentration of p-NBMB employed in

the assay needs to be carefully considered to minimize the dark toxicity from the methylene blue

and the eliminated caged by-product.

In the process of developing activatable PS probes, it was realized that

existing PSs have many other drawbacks in addition to the indiscriminate

phototoxicity. For example, Rose Bengal exhibits photoinduced

deiodination which results in a loss of PS properties25, while porphyrin-based

PSs are difficult to synthesize and exhibit poor water solubility. To this end,

a novel 3-aminocoumarin based PS, called 3AIC (Figure 5.3), was

developed via introduction of iodines at the 6 and 8 positions. In vitro analysis of 3AIC showed

that it can produce singlet oxygen species, while the non-iodinated analogue, 3AC, cannot. The

PS was found to exhibit good photostability, water solubility and could be prepared from

inexpensive commercially available precursors in 2 easy steps with minimal purification. Given

these promising findings, the singlet oxygen quantum yield and the extinction coefficient of 3AIC

should be measured along with the cellular efficacy. In addition, the wavelength of photoexcitation

should ideally be improved via extension of the conjugation in the system such that it absorbs

within the biological optical window.

Figure 5.3. Structure

of the 3AIC.

72

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Appendix A: NMR & Mass Spectrum of Synthesized Compounds

Figure S1. 1H-NMR of guanine salt in DMSO-d6.

79

Figure S2. 1H-NMR of Alkyne O6 Benzyl Guanine in DMSO-d6.

80

Figure S3. 1H-NMR of Alkyne O6 Benzyl Guanine N9 Propylamine BHQ2 in DMSO-d6.

81

Figure S4. 1H-NMR of 3-azidopropan-1-ol in CDCl3.

82

Figure S5. 1H-NMR of 3-azido-N-methylpropan-1-amine in CDCl3. Note, the oil was contaminated with THF and TEA.

83

Figure S6. 1H-NMR of CF3 protected 4-methylamine benzyl alcohol in CDCl3.

84

Figure S7. 1H-NMR of CF3 protected 4-methylamine O6 benzyl guanine in DMSO-d6.

85

Figure S8. 1H-NMR of N-acetylglycine in DMSO-d6.

86

Figure S9. 1H-NMR of 3AIC-Acetate in DMSO-d6.

H2

O

DMSO-

d6

87

Figure S10. 1H-NMR of 3AIC in CDCl3.

CDCl3

88

Figure S11. ESI- mass spectrum of RB-N3 dissolved in methanol.

89

Figure S12. ESI- mass spectrum of probe 1 (P1) dissolved in methanol.

90

Figure S13. ESI+/ESI- mass spectrum of Sarcosine linked pyro A dissolved in methanol.

91

Figure S14. ESI+ mass spectrum of probe 1 no quencher (P1NQ) dissolved in methanol.

92

Figure S15. ESI+ mass spectrum of p-NBMB dissolved in methanol.