Title Mechanism of Degradation of Pharmaceutical
Products and Analogues, and Development of a Novel Fluorescence Technique
for DNA-damage Detection
Adviser Prof. J.C. (Tito) Scaiano
Thesis Committee Prof. Jean Cadet, Dep. de Recherche Fondamentale
sur la Matiere Condensee, Grenoble, France; Prof. Alex Fallis, Dep.
of Chemistry, University of Ottawa; Prof. P. Mayer, at University
of Ottawa; Prof. J.S. Wright, Dep. of Chemistry, Carleton University,
Photochemistry in biological systems has received an
ever-increasing attention in the last few years. This is due to the
many practical applications of the field, examples of which are phototherapy
in cancer treatment, as well as the development of new diagnostic
procedures. Interest is also triggered by the deleterious effects
that sunlight is producing in human body as result of an increase
in the UV portion of the sun spectrum reaching the earth surface.
Damage can be best summarized by skin photosensitization reactions
and radiation induced mutations.
At the time of initiating my graduate studies I was
interested in areas of biological significance where research would
have a direct impact on our quality of life. I thus pursued a molecular
level understanding of the effect of UV radiation in our organism.
I also worked on the application of photochemical techniques for diagnostic
procedures. My graduate research project in photochemistry in biological
systems thus encompassed three very important and interconnected aspects
related to solar radiation and human health: prediction, prevention,
Toxic reactants are a common result of the interaction
of sunlight with pharmaceutical agents transported in the blood system
or applied topically. It was striking to observe that both the anti-inflammatory
agent ketoprofen as well as the hypolipidaemic drug fenofibric acid
were closely related to benzophenone (Figure 1). The latter is the
longest known and best understood model of radical like activity following
In the case of ketoprofen, time resolved experiments
established an efficient singlet state mediated decarboxylation, occurring
within picoseconds, and followed up by protonation of the resulting
carbanion in a few nanoseconds. This fast deactivation ruled out the
involvement of the excited state or any of the subsequent intermediates
in adverse photosensitization effects. A similar situation aroused
for fenofibric acid, which rapidly, (i.e., within hundreds of nanoseconds)
decayed to starting material or photoproducts. Given these fast decays
characterizing the longer lived transients of ketoprofen and fenofibric
acid, oxygen involvement in their deactivation in the living tissues
was predicted not to play a major role. The phototoxicity reported
for these molecules was therefore explained by their efficient generation
of photoproducts. These photoproducts lack a fast deactivation mechanism
following photoexcitation and are prone to undergo Type I reactions
with cellular components, or Type II reactions with oxygen in the
cell, as has been established to be the case with benzophenone.
1. From left to right: structure of benzophenone and the drugs fenofibric
acid and ketoprofen.
The possibility of generating a carbanion within the
duration of a laser pulse was very attractive for mechanistic studies.
Thus ketoprofen photogenerated carbanion grasped our attention as
an exciting substrate to study in order to better understand the chemistry
of carbanions in different systems like water or DMSO. With this in
mind, we examined different ketoprofen derivatives. Upon photoexcitation,
and subsequent decarboxylation, these derivatives were expected to
render their respective carbanions which would in turn react with
water or undergo an intramolecular SN2 or E2 reaction (see
Scheme 1). Every textbook in Organic Chemistry dedicates one or more
chapters to nucleophilic substitution, one of the most versatile reactions
in chemical synthesis, yet not a single absolute rate constant is
known for carbanions. Our work established those values for the first
time, as well as protonation rate constants for these carbanions.
1. Reactions of 1-(3-benzoyl-phenyl)-alkyl carbanions.
We also investigated the capability that naturally occurring
as well as artificially generated protective substrates have to prevent
damage from both solar radiation and its undesired photoproducts.
This lead to preliminary work on new sunscreens based on the novel
concept of isolation of the photoactive ingredient within a chemically
inert, transparent framework, as given by zeolite cages. We also determined
the feasibility of formation of the natural occurring protective pigment
melanin as a result of degradation of its precursors in the presence
of light-elicited toxins. Thus the properties of the adrenaline derived
radical were evaluated, as a case study for the catecholamine group
in general. Absolute rate constants for tert-butoxyl radical scavenging
and triplet benzophenone quenching were reported, and a reactivity
comparison was established with other intermediates involved in the
reaction of melanin formation via catecholamines.
Equally interested in the diagnosis of radiation damage,
we developed a novel technique for the rapid evaluation of DNA damage.
This technique is based on the photophysical behavior of DNA fluorescent
probes. It allows one to determine damage exerted by the phototoxic
agents to the nucleic acid, the genetic information carrier and a
common target of the previously mentioned agents. The rigidity imposed
by the DNA base pairs on intercalating chromophores was exploited
(Figure 2). Its retardation effect on the relaxation of a photoexcited
DNA-stain probe was employed to determine the amounts of DNA existing
as double and single stranded form; which is ultimately an expression
of the damage the DNA has suffered.
2. Schematic representation of dye-dsDNA and dye-ssDNA complexes;
z is the direction of the helix axis. The planes represent the DNA
bases. Following excitation twisting along the methine bridge converts
the molecule into the excited perpendicular singlet state wherefrom
it decays nonradiatively. This internal rotation is far more restricted
in double stranded DNA, as compared to single stranded DNA.
In summary, our work allowed us to establish a systematic
approach for studying the mechanism of degradation of pharmaceutical
products using fast spectroscopy techniques. These studies are essential
to predict (and eventually prevent) the appearance of phototoxic and
photoallergic side effects. An understanding of dye-DNA interactions
was also gained, and employed in the diagnosis of radiation elicited
DNA damage. Preliminary studies also showed the feasibility of elaborating
sunscreens based on the novel concept of encapsulation, which is aimed
at preventing the side effects present in many current sunscreen photoactive