Nanosecond time-resolved resonance Raman (ns-TR3) spectroscopy was used in this thesis to study the photoreduction reactions and the photochemistry of chloro-substituted benzophenone (ClBP) triplets. The 3-chlorobenzophenone (3-ClBP), 4-chlorobenzophenone (4-ClBP) and 4,4′-dichlorobenzophenone (4,4′-diClBP) triplets exhibit similar properties to the parent BP triplet. In isopropyl alcohol (IPA), the hydrogen abstraction reactions were observed for the 3-ClBP, 4-ClBP and 4,4′-diDlBP triplets. The diphenylketyl (DPK) radicals produced from the hydrogen abstraction reactions were observed and the recombination of the DPK and dimethylketyl radicals at the para-position was observed to form a light absorption transient (LAT) species. In MeCN:H2O/1:1 aqueous solutions, these DPK radicals were also observed but with a slower formation rate and the LAT species was produced by reaction with OH radicals. Density functional theory (DFT) calculations were employed to help identify the intermediates seen in the TR3 spectra and to help provide information about the vibrational motions of the molecules examined. The 2-Cl-DPK radical was also observed in the TR3 spectra obtained in an IPA solvent. However, the yield of the 2-Cl-DPK radical and the hydrogen abstraction rate was observed to be significantly lower than that of the other ClBP examined here under the same experimental conditions. The results DFT calculations show that the 2-chloro substituent changes the geometry and the electron density of the molecular orbitals of the BP triplet so that the 2-chloro substituent reduces the hydrogen abstraction ability the triplet state, which is different the hypothesis put forward by some previous studies that an electron-withdrawing group should increase the photoreduction ability of BP derivatives. Norfloxacin (NF) and Enoxacin (EN) are representative derivatives of Fluoroquinolones (FQ). There are four forms of NF and EN and these different forms can coexist in aqueous solutions. The UV-vis absorption and resonance Raman (RR) spectra of NF and EN have been obtained in neat acetonitrile (MeCN), MeCN:HClO4-H2O/1:1 (pH?1), MeCN:H2O/1:1 (pH?7.5) and MeCN:NaOH-H2O/1:1 (pH?13) solutions. The species observed in the spectra are assigned by comparison of the experimental spectra to the DFT calculated spectra and the vibrational modes are also described from the results of the DFT calculations. The absorption spectra of NF and EN obtained in MeCN:H2O/1:1 solutions show that some other species coexist with the tautomeric forms in the neutral aqueous solution. The RR spectra of the tautomeric forms of NF and EN were obtained by subtraction of the RR spectra of the neutral and anionic forms from the RR spectra of NF and EN in neutral aqueous solutions. The results suggest that NF and EN exists in neutral, anionic and tautomeric forms in neutral aqueous solutions. The time dependant DFT calculation results suggest that the fluorine atom has little contribution to the lowest unoccupied molecular orbitals of the different forms of NF and EN. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Nanosecond time-resolved resonance Raman spectroscopy (ns-TR3), nanosecond transient absorption (ns-TA) and femtosecond transient absorption (fs-TA) were utilized to investigate the photochemistry of ketoprofen (KP), ketoprofen-purine dyads, fenofebric acid (FA) in different solutions. For KP, the rate constant and reaction mechanism of KP are strongly dependent on the concentration of water. In neat acetonitrile and acetonitrile-rich solutions (water:acetonitrile?1:1, v:v), KP exhibits mostly benzophenone-like photochemistry to give rise to triplet state which in turn transforms to ketyl radical intermediate by hydrogen abstraction reaction. However, in aqueous solutions with higher water ratios (water:acetonitrile?80%) or acidic solutions, fs-TA studies found that after the irradiation of KP the singlet state will transform into the triplet state with a high efficiency through an intersystem crossing and a triplet state mediated decarboxylation reaction of KP is confirmed in water-rich and acidic solutions as well as the triplet state KP- anion generating a KP carbanion through a decarboxylation reaction. Triplet state ketoprofen (3KP) is firstly observed by ns-TR3 experiments and then excited triplet state intramolecular proton transfer (ESIPT) induces 3KP to facilely undergo the decarboxylation reaction to generate a triplet protonated carbanion biradical (3BCH) species, this observation is also confirmed by the results from density functional theory (DFT) calculations. For solutions with higher water concentrations (such as between 50% and 90% water by volume), the hydrogen abstraction and decarboxylation processes are two competitive pathways with different rate constants. For KP-purine dyads, intramolecular hydrogen abstraction has been proposed to form ketyl-C1 biradical in acetonitrile solvent. Fs-TA study on KP-purine nucleoside dyads reveals that 3KP of cisoid dyads decays faster than 3KP of transoid dyads obtained in acetonitrile-water mixtures. Ns-TR3 experiments and DFT calculations suggest that ketyl-C1 biradical intermediate is generated with a higher efficiency for the 5-KP-dG dyad than for the 5-KP-dA and 5-KPGly-dA dyads. There is no ketyl-C1 biradical observed in ns-TR3 experiments for the 3-KP-dA dyad with transoid structure due to a steric effect. For FA, a solvent dependent photochemistry is observed. A typical nπ* triplet state FA (3FA) is evolved by a high efficient intersystem crossing in acetonitrile-rich solutions and subsequently 3FA promptly abstracts a hydrogen from water molecule to generate a ketyl radical intermediate. In contrast, an inversion of the hydrogen abstraction and decarboxylation reactions of nπ* 3FA is rationalized with the assistance of water molecules when going from acetonitrile-rich to water-rich mixtures. However, in 50% PBS solution, FA carbanion is observed from the picosecond to nanosecond times and the cleavage of FA carbanion gives rise to the enolate 3- anion at later nanosecond delay times. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Femtosecond time-resolved transient absorption (fs-TA), nanosecond time-resolved transient absorption (ns-TA), and nanosecond time-resolved resonance Raman spectroscopy (ns-TR3) methods were used to study the behaviors of the transient intermediates involved in the photophysical and photochemical processes of 3-methylbenzophenone (3-MeBP), 3-(hydroxymethyl)benzophenone (m-BPOH), and 2-(1-hydroxyethyl) 9,10-anthraquinone (2-HEAQ). A particular focus of this work was to study the unusual meta methyl activation reactions of these compounds in water-containing solutions. Density functional theory (DFT) calculations were conducted to help make assignments of the observed experimental transient species and to better understand the reaction mechanisms. First, the photophysical and photochemical reactions of m-BPOH were investigated in selected solvents. In acetonitrile (MeCN) the formation of the triplet state of m-BPOH, (denoted as (m-BPOH)3 ), was detected via an intersystem crossing (ISC). In 2-propanol (IPA), (m-BPOH)3 abstracted a hydrogen atom from the solvent molecule to form an aryl ketyl radical. In an acidic mixed aqueous solution at pH 2, the photoredox reaction appeared to be the predominant reaction. In a more acidic aqueous solution with [H+] =1.0 M, the photoredox reaction faced some competition from the overall photohydration reaction. Second, the photophysical and photochemical reactions of 2-HEAQ in MeCN, IPA, and neutral, acid and basic aqueous solutions were studied. The ISC process of 2-HEAQ took place in MeCN with generation of the triplet excited state species of 2-HEAQ, (2-HEAQ)3. In IPA solvent, (2-HEAQ)3 underwent a hydrogen abstraction with the solvent. A photoredox reaction takes place via an initial protonation process of the AQ group that is followed by deprotonation of the methylene C-H bond in aqueous solutions within a pH range from 2 to 10. Under a stronger acidic aqueous condition with [H+] =1.0 M, the photohydration reaction becomes the major reaction. In strong basic solutions (pH=12) only ISC was observed to take place. The unusual photoredox reaction takes place via protonation of the carbonyl oxygen first followed by deprotonation of the C-H bond in the side chain for both m-BPOH and 2-HEAQ. The protonation of the excited carbonyl oxygen group has been widely studied. On the other hand, the deprotonation of methylene C-H bond is unusual. Therefore, this photoredox reaction for m-BPOH and 2-HEAQ is termed as a meta methyl activation reaction. Third, the photophysical and photochemical reactions of 3-MeBP were explored and compared to those of 4-methylbenzophenone (4-MeBP). This work found that 3-MeBP and 4-MeBP exhibit similar behaviors with m-BPOH and 2-HEAQ in MeCN and IPA. In MeCN, both 3-MeBP and 4-MeBP undergo an efficient ISC process producing triplet excited state species, (3-MeBP)3 and (4-MeBP)3, respectively. In IPA, the (3-MeBP)3 and (4-MeBP)3 intermediates were quenched by the hydrogen abstraction reaction with the solvent. In acidic aqueous solutions (pH  2), the protonated carbonyl oxygen species (3-MeBPH+)3 and (4-(MeBPH+)3 are directly observed by fs-TA spectra. In the case of 4-MeBP, a photohydration is detected and the m-(4-MeBPH2O)3 and o-(4-MeBPH2O)1 species are observed. In contrast, an unusual meta methyl activation reaction is observed for 3-MeBP. In a stronger acid aqueous solution ([H+] =1.0 M) the meta methyl activation reaction becomes the predominant reaction. / published_or_final_version / Chemistry / Doctoral / Doctor of Philosophy

Peptide receptor overexpression in diseased cells and tissues, including carcinomas provides an opportunity to develop therapeutics and imaging agents that selectively bind to such cells and tissues. This dissertation presents tools and processes that can be utilized to target melanocortin and cholecystokinin receptors through multivalent binding. In Chapter 2, improved synthesis and purification methods are described for the production of Eu-chelated probes that serve to evaluate the binding efficacy of multivalent molecules through competition binding assays. Specifically, a xylenol orange-based assay for quantification of unchelated metal ions was used to determine unbound metal ion contamination and the success of metal ion removal. The use of Empore™ chelating disks was determined to be the method of choice for the selective removal of unchelated Eu ions from several Eu-diethylenetriaminepentaacetic acid chelate-peptide conjugates. Applying new synthesis and purification strategies, the TRF probe Eu-DTPA-PEGO-CCK4 targeted to cholecystokinin receptors was synthesized (Chapter 2) and validated via saturation and competition binding assays (Chapter 4) using a HEK293 cell line overexpressing the human cholecystokinin 2 receptor. In Chapter 3, short and efficient syntheses of multivalent molecules targeted to melanocortin receptors based on three commercially available trigonal core scaffolds, phloroglucinol, tripropargylamine, and 1,4,7-triazacyclononane, are described. These constructs were designed to further test the 24 ±5 Å inter-ligand distance suggested in recent literature for multivalent binding to melanocortin receptors. The bioactivities of these compounds were evaluated using a competitive binding assay that employed HEK293 cells engineered to overexpress the human melanocortin 4 receptor. In the course of conducting these bioassays, novel in vitro binding assay protocols were established, which led to high repeatability and robustness of the bioassays compared to previous methods. The divalent molecules exhibited 10- to 30-fold higher levels of inhibition when compared to the corresponding monovalent molecules, consistent with divalent binding. The trivalent molecules were only statistically (~2-fold) better than the divalent molecules, still consistent with divalent binding but inconsistent with trivalent binding. Possible reasons for these behaviors and planned refinements of the multivalent constructs targeting melanocortin receptors based on these scaffolds are discussed in Chapters 3 and 6.

This thesis is an investigation of the photophysical processes that occur in organic photovoltaic blends in the time between light being absorbed and free charges being generated. The purpose of all solar cells is to generate a photocurrent. The free charges, as they flow out of the device, make up the photocurrent, so understanding the processes by which they are created is vitally important to organic photovoltaic research. The main experimental method used was time-resolved fluorescence spectroscopy. This technique was used to probe the exciton population with respect to time for a variety of blends of organic semiconductors, including the high performance photovoltaic materials PCDTBT, PTB7, C71-PCBM and P3HT. The main goal of the work was to characterise the exciton diffusion lengths of these materials by developing a technique called volume quenching. Volume quenching involves blending a small quantity of quenching material into a thin film of semiconducting material. These introduced quenching sites render excitons unemissive on contact. Thus, from the drop in fluorescence compared with the ‘unquenched' material, it was possible to work out what proportion of the initial excitons have encountered a quenching site in the blends. The results can then be fitted to quantify how diffusive the excitons are -i.e. how far they move. By looking at the rate constant of the quenching process and how it varies with respect to time, quencher concentration and quencher type, it was possible to generate a wealth of additional information, not just about exciton diffusion, but about all the inter-related processes that contribute to exciton harvesting. These processes included the measurement of long-range energy transfer from the donor to the acceptor, electron transfer at the interface with the acceptor and the understanding of nanomorphology of donor-acceptor heterojunctions.

Title: Spectroscopic Study of Singlet Oxygen in Cells and Model Systems Author: Marek Scholz Department: Department of Chemical Physics and Optics Supervisor: doc. RNDr. Roman Dědic, Ph.D., KChFO Abstract: Singlet oxygen (1O2), the first excited state of molecular oxygen, plays many important roles in nature and technology. The work is aimed at development of novel methods for monitoring of 1O2 in cells and other biological samples. Two main ap- proaches were employed: direct detection of the very weak near-infrared phospho- rescence of 1O2, and detection of Singlet Oxygen-Feedback Delayed Fluorescence (SOFDF), which is the emission from the photosensitizer induced by energy transfer from 1O2. The first part of the thesis introduces the basic concepts of photophysics and photochemistry of 1O2: its generation, deactivation, applications, and overview of detection methods. The second part presents the experimental results. Wide-field mi- crospectroscopic detection of 1O2 phosphorescence enabled us to acquire 1O2-based images and near-infrared spectra from single cells incubated with photosensitizers. However, the direct detection suffers from the inherently very low phosphorescence quantum yield. It is shown that SOFDF may overcome this problem and become a promising alternative tool for studies of 1O2 and...

Deoxyribonucleic acid (DNA) forms the basis of all known living organisms. Despite the essential role played by DNA, its dynamic system and functional behaviour are still not completely understood. The work presented in this thesis aims to explore the structural dynamics of DNA systems, using fluorescence-based approaches, and to attempt to develop a technique for the measurement of fluorescence decays of biological molecules on the ultrafast (femtosecond) timescale. Absorption of UV radiation by DNA is known to lead to mutations and damage to DNA structure and functionality. For the majority of absorbed photons, the excitation energy dissipates harmlessly as heat, but in some instances this energy transfers to regions of DNA that are more susceptible to damage. 2-Aminopurine (2AP), a fluorescent analogue of the native DNA base adenine, can be incorporated into DNA with minimal perturbation to the DNA structure, and can be used to investigate inter-base electronic energy transfer. By selectively exciting the native DNA base in 2AP-containing dinucleotides and utilising 2AP fluorescence as an energy acceptor, the mechanism of electronic energy transfer has been investigated. Analysis of the resulting fluorescence lifetimes of 2AP has revealed that energy transfer preferentially excites conformations in which the bases are highly stacked, and the fluorescence of 2AP is highly quenched. This has led to a re-evaluation of energy transfer efficiencies between the natural bases and 2AP, and has shown that transfer efficiencies cannot be determined correctly from steady-state fluorescence measurements. To investigate the influence of base dynamics on the quenching of 2AP fluorescence in DNA, time-resolved fluorescence measurements were carried out on 2AP-containing systems in frozen solution at 77 K. These studies included dinucleotides, single–strand oligonucleotides and their corresponding duplexes. In all cases, comparison of the fluorescence decay parameters measured at room temperature with those measured at 77 K showed that elimination of base dynamics prevented rapid quenching, on the 10s of ps timescale or faster, although quenching on the 100s of ps timescale persisted for 2AP in single strands and duplexes. The multi-exponential fluorescence decay of 2AP in DNA and its high sensitivity to local environment is commonly exploited to investigate DNA-enzyme interactions. Transposases are enzymes involved in the movement of sections of DNA (transposons) within the genome. The Mos1 transposase catalyses the movement of a transposon via a cut-and-paste mechanism involving several intermediate complexes. Understanding the complex mechanism by which the transposase can remove and insert a section of DNA would allow these enzymes to be used as biomolecular tools. The structure of the intermediate Mos1 strand-transfer complex (STC) has been investigated by incorporating 2AP into several regions of the transposon and analysing the fluorescence decay. The involvement of a base-flipping-like mechanism has been identified in the mechanism of strand transfer for the Mos1 transposon. The time-resolved fluorescence measurements performed in this thesis are limited to time resolution of ~20 ps and longer using TSCPC. However, an abundance of photophysical events in DNA occur on the femtosecond timescale. Development of a methodology utilising fluorescence gating techniques (such as sum-frequency generation or diffraction from a transient grating) have been attempted, in order to construct an experimental system that enables the broadband detection of ultrafast fluorescence decays. Despite the lack of immediate success in recording the fluorescence decay from a sample, due to technical issues and time-constraints, initial characterisation of the set-up was performed and the prospect of broadband detection was demonstrated. Overall, this thesis gives insight into some of the dynamic processes taking place in DNA and presents work performed to develop a system that would allow the extension of these studies to processes occurring on the fs timescale.

abstract: Photosystem II (PSII) is a large protein-cofactor complex. The first step in photosynthesis involves the harvesting of light energy from the sun by the antenna (made of pigments) of the PSII trans-membrane complex. The harvested excitation energy is transferred from the antenna complex to the reaction center of the PSII, which leads to a light-driven charge separation event, from water to plastoquinone. This phenomenal process has been producing the oxygen that maintains the oxygenic environment of our planet for the past 2.5 billion years. The oxygen molecule formation involves the light-driven extraction of 4 electrons and protons from two water molecules through a multistep reaction, in which the Oxygen Evolving Center (OEC) of PSII cycles through 5 different oxidation states, S0 to S4. Unraveling the water-splitting mechanism remains as a grant challenge in the field of photosynthesis research. This requires the development of an entirely new capability, the ability to produce molecular movies. This dissertation advances a novel technique, Serial Femtosecond X-ray crystallography (SFX), into a new realm whereby such time-resolved molecular movies may be attained. The ultimate goal is to make a “molecular movie” that reveals the dynamics of the water splitting mechanism using time-resolved SFX (TRSFX) experiments and the uniquely enabling features of X-ray Free-Electron Laser (XFEL) for the study of biological processes. This thesis presents the development of SFX techniques, including development of new methods to analyze millions of diffraction patterns (~100 terabytes of data per XFEL experiment) with the goal of solving the X-ray structures in different transition states. ii The research comprises significant advancements to XFEL software packages (e.g., Cheetah and CrystFEL). Initially these programs could evaluate only 8-10% of all the data acquired successfully. This research demonstrates that with manual optimizations, the evaluation success rate was enhanced to 40-50%. These improvements have enabled TR-SFX, for the first time, to examine the double excited state (S3) of PSII at 5.5-Å. This breakthrough demonstrated the first indication of conformational changes between the ground (S1) and the double-excited (S3) states, a result fully consistent with theoretical predictions. The power of the TR-SFX technique was further demonstrated with proof-of principle experiments on Photoactive Yellow Protein (PYP) micro-crystals that high temporal (10-ns) and spatial (1.5-Å) resolution structures could be achieved. In summary, this dissertation research heralds the development of the TR-SFX technique, protocols, and associated data analysis methods that will usher into practice a new era in structural biology for the recording of ‘molecular movies’ of any biomolecular process. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2015

abstract: Time-resolved serial femtosecond crystallography is an emerging method that allows for structural discovery to be performed on biomacromolecules during their dynamic trajectory through a reaction pathway after activation. This is performed by triggering a reaction on an ensemble of molecules in nano- or microcrystals and then using femtosecond X-ray laser pulses produced by an X-ray free electron laser to collect near-instantaneous data on the crystal. A full data set can be collected by merging a sufficient number of these patterns together and multiple data sets can be collected at different points along the reaction pathway by manipulating the delay time between reaction initiation and the probing X-rays. In this way, these ‘snapshot’ structures can be viewed in series to make a molecular movie, allowing for atomic visualization of a molecule in action and, thereby, a structural basis for the mechanism and function of a given biomacromolecule. This dissertation presents results towards this end, including the successful implementations of the first diffusive mixing chemoactivated reactions and ultrafast dynamics in the femtosecond regime. The primary focus is on photosynthetic membrane proteins and enzymatic drug targets, in pursuit of strategies for sustainable energy and medical advancement by gaining understanding of the structure-function relationships evolved in nature. In particular, photosystem I, photosystem II, the complex of photosystem I and ferredoxin, and 3-deoxy-D-manno-2-octulosonate-8-phosphate synthase are reported on, from purification and isolation, to crystallogenesis, to experimental design and data collection and subsequent interpretation of results and novel insights gained. / Dissertation/Thesis / Doctoral Dissertation Chemistry 2018

Abstract The development of commercially available assays from the measurement of enzyme activity to mass concentrations of proteins, especially the assays of cardiac troponin I and T, has been the most important innovation in the field of cardiovascular laboratory diagnostics over the decade. The availability of a simple, rapid test using whole blood to facilitate processing and to reduce the turnaround time could improve the management of patients presenting with chest pain. The aim of this study was to evaluate the analytical and clinical performance of a new time-resolved fluorometry-based immunology technology using the cardiac marker and high-sensitivity C-reactive protein assays. In addition, the use of high-sensitivity C-reactive protein assay for the investigation of patients with acute atrial fibrillation and the influence of heparin for cardiac marker assays were studied. The levels of precision attained with pooled serum and plasma samples and control materials were acceptable. The assays were found to be linear within the ranges tested. The correlation coefficient between the Innotrac Aio! 1st generation cTnI and Abbott AxSYM cTnI assays was 0.960, and the slope was 0.07. The correlations between the 2nd generation Innotrac Aio!, Access AccuTnI and Abbott AxSYM assays were good, but there were biases between the methods. The correlation coefficients between the Innotrac Aio! and Abbott AxSYM CK-MB and myoglobin assays were 0.995 and 0.971, respectively, but the Innotrac Aio! CK-MB assay yielded about 9% higher values than the Abbott assay. The correlations between Innotrac Aio! usCRP and Cobas Integra CRP latex and between Innotrac Aio! usCRP and Hitachi CRP (Latex ) HS were good. Furthermore, the sample material correlation studies showed no significant differences when the Innotrac Aio! System was used. However, the mean Abbott AxSYM CK-MB values and the cTnI values for heparin plasma samples were 17% higher and about 15% lower than for serum samples, respectively. In the investigation of CRP levels in patients with acute atrial fibrillation CRP tended to be higher in the patients with acute FA, and there was a positive correlation between the concentrations of CRP and IL-6. The results demonstrate the excellent analytical performance of the Innotrac Aio! 2nd generation cTnI, myoglobin, CK-MB and usCRP assays, and all the matrices, including serum, plasma and whole blood, are suitable sample matrices to be used with these methods without further standardization.