Excited state studies of pyramidine bases and radiosensitizing drugs by laser flash photolysis

The mechanism whereby radiosensitizing drugs act in the radio- therapeutic treatment of cancer is yet to be fully elucidated. The prevailing current view (the so-called charge sequestration model) is that cancer cell death is initiated by charge separation induced by the ionising radiation, yielding radical anions (of thymine) and cations (of guanine) in the DNA polymer chain. The radiosensitizer, by virtue of its electron-affinic properties, removes the electron from the (thymine) radical anion, thereby preventing charge recombination and allowing the radical cation to 'fix' the damage via Secondary reactions. To date, most efforts to verify this model have involved the observation of various DNA radical ions by electron spin resonance and pulse radiolysis techniques. However, excited states of DNA or the drug, as well as radical ions, may be involved in the sensitizing action, and there have been a variety of laser flash photolysis studies of reaction between the excited radiosensitizer and ground state nucleic acid bases. The main body of this thesis (Chapter 3) has been to determine, using 249 nm laser flash photolysis, whether reaction in solution occurs between triplet excited nucleic acid bases (in particular thymine and uracil) and (ground state) radiosensitizing drugs. (Such a study has only recently been made feasible by the development of powerful ultraviolet pulsed lasers which are able to produce measurable concentrations of these triplet states in solution; even so, monitoring systems are extended to their limits of detection.) The triplet states of thymine and uracil (in acetonitrile) were quenched by a variety of electron acceptor molecules, including radiosensitizing drugs. The quenching kinetics correlated with the electron affinities of the electron acceptors according to the Weller equation for excited state electron transfer. This constitutes positive evidence that triplet DNA bases can produce the radical cations which are presumed to lead to cell death. Further, in certain cases, the radical anions of the acceptors were observed optically and their yields measured. In Chapter 4 are presented studies of the triplet states of two radiosensitizing drugs, metronidazole and misonidazole (principally by laser flash photolysis), including measurements of their triplet energies. The reduction potentials of these drugs (in acetonitrile) were determined by three methods, which gave comparable results. Attempts were made to develop a fluorescent probe which could be used to measure intracellular concentrations of radiosensitizing drugs. In Chapter 5, the efficient quenching of both excited uranyl ion and triplet benzophenone by nucleic acid bases is detailed. From the results it is concluded that for uranyl ion, the mode of quenching is by an exciplex or reversible charge transfer mechanism, while for triplet benzophenone, chemical quenching occurs. The appendices are mainly concerned with various computer-based techniques developed for this study. An original method is described for analysing oscilloscope transient decays by photographing the oscilloscope screen using a video camera and transferring and digitising the resulting image into a micracomputer screen ram area, where it is manipulated to yield the transient decay constant. Also given is a computer program developed to enable optimum fitting of quenching data to the Weller equation, when the donor oxidation potential is unknown. Finally, evidence for the triplet state of 5-nitroindole is reported. A part of this work has been published, viz. "Electron-transfer Quenching of Triplet State Thymine and Uracil", T.J. Kemp, A.W. Parker, and P. Hardman, J. Chem. Soc, Chem. Commun., 1985, 1377

Theoretical study on chemical bonds, energy transfers, and reaction dynamics in excited states

制度:新 ; 文部省報告番号:甲2512号 ; 学位の種類:博士(理学) ; 授与年月日:2007/12/13 ; 早大学位記番号:新463

Mechanism of action studies on the FR-9000482 class of antitumor antibiotics

1997 Summer.Includes bibliographical references.The interactions of members of the FR-900482 class of antitumor antibiotic agents with DNA has been examined. Importantly, the first in vitro demonstration of nucleic acid interstrand cross-linking has been reported and the DNA base pair sequence specificity of the cross-linking event has been elucidated. These agents demonstrate a high degree of selectivity for 5'-CG-3' sequences of DNA. As such, bio-mechanistic analogy between these compounds and the clinically employed compound Mitomycin Chas been shown. Efforts have also examined extensively the ability of these agents to give rise to orientation isomers of each respective cross-link and their different properties. DNA-protein cross-linking by these agents has also been examined. A sequence-specific DNA-peptide binding motif has been identified which undergoes drug-mediated DNA-protein cross-linking. This is the first reported instance of a mitosene based-minor groove DNA-protein cross-link event. Significantly, the motif examined is characteristic of tissues which bear striking similarity to those of cancerous cell lines

Fotosensibilización y fototerapia con furocumarinas: Un estudio mecano-cuántico.

RESUMEN La Fototerapia es uno de los campos de la Medicina que ha experimentado un mayor desarrollo en las últimas décadas, ampliándose cada vez más el rango de enfermedades que pueden ser tratadas de esta forma. Quizás esta terapia representa como ninguna otra la combinación entre la Física (radiación electromagnética) y la Química (el fármaco empleado o fotosensibilizador) para combatir enfermedades. El objeto de la presente Tesis Doctoral es el estudio teórico de compuestos empleados como fármacos en la denominada terapia PUVA, denominados furocumarinas. El estudio teórico químico-cuántico de los estados electrónicos excitados de estas moléculas permite hallar alguna de las claves del mecanismo de acción fototerapéutica, que, según la literatura, se basa en la fotoconjugación con las bases pirimidínicas del ADN por un lado y en la reacción con el oxígeno molecular del medio celular produciendo oxígeno singlete tóxico por otro (terapia fotodinámica). Nuestro estudio del mecanismo tiene como principales objetivos proponer mejores fotosensibilizadores y desentrañar el mecanismo fotoquímico subyacente en este tipo de fotoquimioterapia. Inicialmente hemos centrado nuestros cálculos, mediante la metodología CASPT2//CASSCF, en el estudio de los estados excitados singletes y tripletes cuya participación en los procesos fotoquímicos radiativos y no radiativos es más probable a fin de estudiar las principales propiedades fotofísicas de los compuestos (espectros de absorción y emisión, fuerzas de oscilador, momentos dipolares, tiempos de emisión radiativa, orígenes de banda, acoplamiento espín-órbita), en un primer paso para racionalizar su mecanismo de actuación. Una vez analizado si se puebla eficientemente el estado excitado triplete (protagonista de la acción fotosensibilizadora) de cada molécula, estudiaremos cómo interaccionan estos fármacos con la timina del ADN y con el oxígeno molecular. Por último, se ha estudiado la formación de los dímeros de timina, una de las lesiones más frecuentes en el ADN, y cuya formación sigue un mecanismo similar a la correspondiente a la interacción entre las furocumarinas y la propia timina. Para ello será clave el análisis de las hipersuperficies de energía potencial de los estados implicados en un proceso dado, así como los cruces entre las mismas y las barreras energéticas para pasar de una región a otra de la hipersuperficie. De hecho, la Fotofísica y la Fotoquímica modernas están basadas en el estudio de las hipersuperficies, las cuales pueden ser consideradas como el terreno de juego donde tienen lugar los procesos físicos y químicos. __________________________________________________________________________________________________This thesis is focused on studying the photochemical behaviour of the furocoumarins in order to propose the best photosensitizer. Firstly the photophysics of psoralen, the parent molecule, will be studied. Afterwards how the lowest triplet excited state is populated will be analized, in both psoralen and thymine, since this state is the responsible for the photosensitizing action. The next step is studying the photocycloaddition between psoralen and thymine that culminates in the formation of monoadducts and diadducts in DNA, which is the key point in the photosensitizing ability of these compounds. A parallel study of other furocoumarins (8-MOP, 5-MOP, TMP, khellin and 3-CPS) will be carried out in order to rationalize which is the best drug from a quantum-chemical viewpoint. We will consider the other side of PUVA therapy as well: the interaction of furocoumarins with molecular oxygen through energy transfer to yield singlet oxygen, which is a strong electrophilic species that reacts with some components of the cellular membrane causing cell death by apoptosis. Finally, the properties of thymine dimers, formed via another sort of photocycloaddition reaction, will be studied. The overwhelming majority of the calculations presented here will be carried out with the CASPT2//CASSCF methodology, that is, energies at CASPT2 level and geometries at CASSCF level. Specifically, we will compute the lowest-lying singlet and triplet excited states, in order to rationalize the photophysical (electronic energies, oscillator strengths, dipole moments, radiative lifetimes, band origins, spin-orbit coupling) and photochemical properties (photocycloaddition, energy transfer) of furocoumarins

Estudio teórico de procesos fotoinducidos en sistemas biológicos

En la presente Tesis Doctoral se investiga, desde un punto de vista teórico, procesos de transferencia de carga y/o energía inducida por la luz en distintos sistemas biológicos. En concreto se estudian tres procesos paradigmáticos: la transferencia de carga en la fotosíntesis, la fotofísica y fotorreactividad de las bases nitrogenadas del DNA y el proceso de fotoisomerización del cromóforo de la Rodopsina, desencadenante de la visión. La metodología empleada para la realización de estos estudios es principalmente la CASPT2//CASSCF, una herramienta químico-cuántica especialmente adecuada para el tratamiento de los estados moleculares excitados, debido a su carácter multiconfiguracional.This Doctoral Thesis investigates, from a theoretical point of view, charge and energy transfer processes in biological systems that are induced by light. In particular, three paradigmatic processes are studied: the charge transfer in photosynthesis, photophysics and photoreactivity of DNA nucleobases and the photoisomerization of the chromophore of Rhodopsin, which is the first step in the vision process. These studies has been carried out by using CASPT2//CASSCF methodology, a quantum chemistry tool very useful for the study of excited molecular states due to their multiconfigurational character

Multireference approaches for excited states of molecules

Understanding the properties of electronically excited states is a challenging task that becomes increasingly important for numerous applications in chemistry, molecular physics, molecular biology, and materials science. A substantial impact is exerted by the fascinating progress in time-resolved spectroscopy, which leads to a strongly growing demand for theoretical methods to describe the characteristic features of excited states accurately. Whereas for electronic ground state problems of stable molecules the quantum chemical methodology is now so well developed that informed nonexperts can use it efficiently, the situation is entirely different concerning the investigation of excited states. This review is devoted to a specific class of approaches, usually denoted as multireference (MR) methods, the generality of which is needed for solving many spectroscopic or photodynamical problems. However, the understanding and proper application of these MR methods is often found to be difficult due to their complexity and their computational cost. The purpose of this review is to provide an overview of the most important facts about the different theoretical approaches available and to present by means of a collection of characteristic examples useful information, which can guide the reader in performing their own applications

Liquid chromatography-mass spectrometry analysis of compounds from complex matrices

The aim of the work was to establish two methods for the analysis of 4 thyroid hormones and 18 plant toxins by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using electrospray ionisation (ESI). The developed and validated methods were then applied to samples, with a range of complex matrices, namely serum, simulated gastro-intestinal fluid, tablet formulation and plants, with sample preparation methods developed and validated for each. Thyroid disorders, hypothyroidism and hyperthyroidism, are a common medical condition with diagnosis obtained via the analysis of thyroid hormones present within serum samples. Currently within the NHS analysis is performed using electrochemiluminescence to measure thyroid stimulating hormone, thyroxine and triiodothyronine. However, these methods have limited sensitivity and inaccuracies of 30% at the limit of detection. Therefore, diagnosis of hypothyroidism can often be missed with multiple medical visits being required. The developed method allows for the detection and qualification of 4 thyroid hormones, thyroxine, triiodothyronine, reverse triiodothyronine and diiodothyronine, using low resolution and high resolution mass spectrometry. The developed method showed significant improvements over the current NHS method with LOQs of 0.990 and precision of <3.8 %. A direct comparison between the development method and the current clinical method showed that the result obtained were comparable in terms of thyroid hormone levels detected. Allowing for LC-MS/MS to be a suitable, more reliable alternative for the detection and quantification of thyroid hormones. Plant toxins are a common cause of accidental poisoning with a range of symptoms including burns, nausea, convulsions, paralysis and even death. Many of the plants analysed in this study can be purchased from garden centres and online nurseries with little to no safety information provided. A method was developed for the analysis of 18 plant toxins, atropine, aconitine, capsaicin, cathinone, colchicine, coumarin, digitoxin, digoxin, hellebrin, 5-methoxypsoralen, 8-methpxypsoralen, psoralen, salvinorin A, scopolamine, solanine, strychnine, thujone and veratridine, based upon the plant availability. The developed method showed good linearity, r2 > 0.9952 and precision of <10.7 %. The analysis of the plant materials showed toxins consistent with information obtained from the head gardener at Alnwick Gardens, which holds the largest poison garden in the UK. A risk assessment for ingestion and dermal exposure was compared with the LD50 and it was found that Colchicum autumnale posed the greatest risk via ingestion with Atropa belladonna also posing significant risk via ingestion. The dermal risk was minimal; however, care should be taken in the case of dermal contact due to the presence of phototoxins present in plant such as Heracleum mantegazzianum

The Double Helix in Motion: New Insights into Sequence-specific, Functional DNA Dynamics Using NMR Spectroscopy

DNA is a highly flexible molecule that undergoes a variety of structural transitions in response to cellular cues. Sequence-directed variations in the canonical double helix structure that retain Watson-Crick base-pairing play important roles in DNA recognition, topology, and nucleosome positioning. Here, we use NMR relaxation methods to study sequence-directed dynamics occurring at picosecond to millisecond timescales in variable size DNA duplexes. Traditionally, atomic-level spin relaxation studies of DNA dynamics have been limited to short duplexes, in which sensitivity to biologically relevant nanosecond fluctuations is often inadequate. We introduce a method for preparing residue-specific 13C/15N-labeled elongated DNA along with a strategy for establishing resonance assignments and apply it towards probing fast inter-helical bending motions induced by an adenine tract. Our results suggest the presence of elevated A-tract independent end-fraying and/or bending internal nanosecond motions, which evade detection in short constructs and that penetrate deep within the helix and gradually fade away towards its interior. By studying picosecond-nanosecond dynamics in short DNA dodecamers with variable length A-tracts, we discover that A-tracts are relatively rigid and can modulate the flexibility of their junctions in a length-dependent manner. We identify the presence of large-amplitude deoxyribose internal motions in CA/TG and CG steps placed in different sequences that likely represent rapid sugar repuckering. Moreover, by using NMR relaxation dispersion in concert with steered molecular dynamics simulations, we observe transient sequence-specific excursions away from Watson-Crick base-pairing at CA/TG and TA steps inside DNA dodecamers towards low-populated and short-lived A•T and G•C Hoogsteen base pairs. We show that their populations and lifetimes can be modulated by environmental factors like acidity, monovalent and divalent ions as well as intrinsic sequence and chemical modifications. The observation of Hoogsteen base pairs in duplexes specifically bound to transcription factors and in damaged sites implies that the DNA double helix intrinsically codes for excited state Hoogsteen base pairs as a means of expanding its structural complexity beyond Watson-Crick base-pairing. The methods presented here provide a new route for characterizing transient nucleic acid structures, which we predict will be abundant in the genome and constitute a second transient layer of the genetic code.Ph.D.Chemical BiologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/89648/1/nikolove_1.pd

Molecular Dynamics Study of Supercoiled DNA Minicircles Tightly Bent and Supercoiled DNA in Atomistic Resolution

Towards the complete understanding of the DNA response to superhelical stress, sequence dependence structural disruptions on the ~100 base pairs supercoiled DNA minicircles were examined through a series of atomistic MD simulations. The results showed the effects from some subtle structural characteristics of DNA on defect formation, including flexibility at base pair step level and anisotropy, whose dynamic information are available only from atomistic MD simulations. For longer supercoiled DNA minicircles (240-340 bp), the molecules adapt into their writhed conformations. Writhe can be calculated by a Gauss’ integral performed along the DNA central axis path. A new mathematical definition for the DNA central axis path was developed for the more exact writhe calculation. Finally, atomistic representation of supercoiled 336 base pairs minicircles was provided by fitting the DNA structure obtained by explicitly solvated MD simulations into the density maps from electron cryo-tomography. Structural data were analysed and provided a decent explanation for the mechanism of the sequence specific binding of the enzyme topoisomerase 1B onto the negatively supercoiled DNA

Molecular Photochemistry

There have been various comprehensive and stand-alone text books on the introduction to Molecular Photochemistry which provide crystal clear concepts on fundamental issues. This book entitled "Molecular Photochemistry - Various Aspects" presents various advanced topics that inherently utilizes those core concepts/techniques to various advanced fields of photochemistry and are generally not available. The purpose of publication of this book is actually an effort to bring many such important topics clubbed together. The goal of this book is to familiarize both research scholars and post graduate students with recent advancement in various fields related to Photochemistry. The book is broadly divided in five parts: the photochemistry I) in solution, II) of metal oxides, III) in biology, IV) the computational aspects and V) applications. Each part provides unique aspect of photochemistry. These exciting chapters clearly indicate that the future of photochemistry like in any other burgeoning field is more exciting than the past