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A study has been made of a novel fluorescent and phosphorescent label 6-coumarinsulphonyl chloride (6-CSCl) based an the coumarin nucleus with a reactive group similar to dansyl chloride. The synthesis, analysis, derivatisation and application of the label to certain analytical problems have been studied. The label was obtained in a high state of purity by a straightforward sulphonation of the counarin nucleus. Characterisation of the label was carried out using mass spectrometry, NMR and IR, its purity was verified by elemental analysis. The label was found to be highly stable and react at room temperature with phenolic and amino groups. The luminescence properties of the label and its derivatives were investigated both at room temperature and at 77K. The room temperature fluorescence showed a strong pH dependence which was not observed at 77K. Environmental effects on the fluorescence properties of the derivatives at room temperature were investigated. Linearity range, quantum yields and the detection limits of the derivatives were determined. The optimum derivatisation conditions for various compounds were determined by HPLC. The analytical applications of the label were investigated by studying the behaviour of the derivatives in HPLC and TLC systems. In the area of HPLC, both pre-c6lumn and post-column derivatisation were attempted. The ion-pair pre-column derivatisation technique was found to be more suitable for this work. TLC analysis was performed on both normal phase and HPTLC plates. The separated components were detected using absorbance and phosphorimetry. Synthesis of the labels with substituents at the 4 and 7 position of the coumarin nucleus were carried out. The behaviour of the modified labels and their derivatives were compared with the original label and its derivatives.

Sweet clover has become increasingly important as a forage and green manure crop. The main objection in recent years to sweet clover is its coumarin content, identified by a sweet odor and bitter taste, making it less desirable to farm animals. When sweet clover hay spoils, the coumarin is converted into dicoumarol which is toxic to animals, especially to ruminants. When ingested this may cause internal and/or external hemorrhages.

<p>The ability to manipulate the coordination chemistry of metal ions has significant ramifications for the study and treatment of metal-related health concerns, including iron overload, UV skin damage, and microbial infection among many other conditions. To address this concern, chelating agents that change their metal binding characteristics in response to external stimuli have been synthesized and characterized by several spectroscopic and chromatographic analytical methods. The primary stimuli of interest for this work are light and hydrogen peroxide.</p><p>Herein we report the previously unrecognized photochemistry of aroylhydrazone metal chelator ((E)-N&#8242;-[1-(2-hydroxyphenyl)ethyliden]isonicotinoylhydrazide) (HAPI) and its relation to HAPI metal binding properties. Based on promising initial results, a series of HAPI analogues was prepared to probe the structure-function relationships of aroylhydrazone photochemistry. These efforts elucidate the tunable nature of several aroylhydrazone photoswitching properties.</p><p>Ongoing efforts in this laboratory seek to develop compounds called prochelators that exhibit a switch from low to high metal binding affinity upon activation by a stimulus of interest. In this context, we present new strategies to install multiple desired functions into a single structure. The prochelator 2-((E)-1-(2-isonicotinoylhydrazono)ethyl)phenyl (E)-3-(2,4-dihydroxyphenyl)acrylate (PC-HAPI) is masked with a photolabile trans-cinnamic acid protecting group that releases umbelliferone, a UV-absorbing, antioxidant coumarin along with a chelating agent upon UV irradiation. In addition to the antioxidant effects of the coumarin, the released chelator (HAPI) inhibits metal-catalyzed production of damaging reactive oxygen species. Finally a peroxide-sensitive prochelator quinolin-8-yl (Z)-3-(4-hydroxy-2-((4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)benzyl)oxy)phenyl)acrylate (BCQ) has been prepared using a novel synthetic route for functionalized cis-cinnamate esters. BCQ uses a novel masking strategy to trigger a 90-fold increase in fluorescence emission, along with the release of a desired chelator, in the presence of hydrogen peroxide.</p> / Dissertation

Chemical labeling is an important tool for understanding protein structure and function. Biological research often requires the use of molecular labels that are covalently attached to facilitate detection or purification of the labeled protein and its binding partners. Although the number of probes have been developed for labeling of specific residues of proteins is substantial, there is still a need for new reagents with better reactivity, and selectivity. Moreover, these chemical probes should be able to label the protein of interest under mild biologically relevant conditions. Aryl diazonium salts have been utilized for selective modification of tyrosine residues. However, most diazonium compounds need to be generated in situ under strongly acidic conditions due to their instability1. Our group has previously shown that triazabutadienes can be used as precursors that can generate diazonium under mild acidification2 or photo-irradiation3. Current reported systems for bioconjugation of tyrosine require an additional step for fluorescent labeling4. To address this issue and reduce background fluorescence that is associated with fluorescent labeling, coumarin triazabutadiene-based fluorogenic probes were synthesized and tested for tyrosine specific labeling of proteins under mild acidic condition or photo-irradiation. Furthermore, a coumarin triazabutadiene-based cross-linker was synthesized with an azide functionality that can be used to attached the coumarin triazabutadiene warhead onto the surface of a protein. Upon the activation of the triazabutadiene group, by light or lowering the pH, this system can generate a coumarin diazonium salt on the surface of the protein. Such a system can find application in the study of protein-protein interactions and virus-protein interactions. A cyclooctyne triazabutadiene was synthesized to attach a cyclooctyne group on the tyrosine residues of proteins in biologically relevant pH, and 3-azido 7-hydroxy coumarin was made as a fluorogenic partner of the cyclooctyne triazabutadiene. It was demonstrated that this system can label tyrosine residue followed by a copper-free click reaction with the azido coumarin fluorophore. This system has been tested on model proteins and can be consider as one the first fluorogenic triazabutadiene systems that can be utilized for labeling of tyrosine under mild conditions. In conclusion, this dissertation demonstrates progress in developing fluorescent and fluorogenic triazabutadienes systems for labeling of tyrosine residues of proteins as well as fluorophore triazabutadiene cross-linker that can be used for studying protein-protein interaction, and virus-protein interactions. These systems offer a convenient tool to those wishing to study proteins, protein-protein interactions, and virus-protein interactions.

Abstract Cytochrome P450 2A6 (CYP2A6) is involved in the 7-hydroxylation of coumarin, C-oxidation of nicotine, and the metabolism of tobacco specific nitrosamines. Initially in 1995 Fernandez-Salguero et al. reported a genotyping method for three alleles: CYP2A6*1 (wild-type), CYP2A6*2 (variant 1), and CYP2A6*3 (variant 2). Later studies presented in this thesis indicated that the original genotyping method produces erroneous results for the CYP2A6*3 allele due to unspecific PCR conditions and previously unknown CYP2A6*1B allele. Furthermore, the CYP2A6*2 allele genotyping caused erroneous genotypes (CYP2A6*2/*2 was misclassified as CYP2A6*1/*2). In this work, new PCR based genotyping methods were developed for CYP2A6*2 and for several new alleles (CYP2A6*1B, CYP2A6*4A/*4D and CYP2A6*5). In population-based studies, the deletion alleles (pooled as CYP2A6*4) turned out to be more prevalent among Asians (15.1%) than Caucasians (0.5%). The frequencies of the other inactive alleles varied within 0–3% in both populations. Asians totally lacked the CYP2A6*2 allele, whereas Caucasians lacked the CYP2A6*5 allele. The frequencies of two wild-type alleles, CYP2A6*1A and CYP2A6*1B alleles were 66.5% and 30.0% in Caucasians, and 43.2% and 40.6% in Asians, respectively. Correlation studies between the phenotype, as tested by the administration of coumarin, and the genotype demonstrated that individuals with the CYP2A6*2/*2 genotype were totally defective, while CYP2A6*1/*2 subjects exhibited intermediate and CYP2A6*1/*1 subjects full capablility of producing 7-hydroxycoumarin. Upon phenotyping with nicotine, individuals with the CYP2A6*1/*2 or CYP2A6*1/*4 genotype were shown to have a lower enzyme activity (one fourth of the normal activity), compared to those with the CYP2A6*1/*1 genotype. Defective CYP2A6 activity has been hypothesised to reduce the risk of environmentally (especially tobacco smoke) induced diseases either by decreasing production of genotoxic metabolites or by preventing addiction to tobacco smoking. However, in our case-control studies on Spanish patients with liver cirrhosis (n = 83) and liver cancer (n = 90) and their controls (n = 237) no significant association between the CYP2A6 genotypes and disease proneness was found. The odds ratio (OR) for developing liver cancer was was 1.4 (95% confidence interval [CI] 0.5–3.7) for genotypes containing at least one CYP2A6*2 allele. For liver cancer the respective OR was 1.3 (95% CI 0.4–4.5). Similarly, no statistically association between CYP2A6 alleles and the risk of lung cancer was observed in our Finnish study population cinsisting of 177 cases and 1089 controls; the OR for combined CYP2A6 variant allele containing genotypes (CYP2A6*1/*2 and CYP2A6*1/*4) was 1.19 (95% CI 0.56–2.45). Our studies therefore do not indicate any major modifying role for the CYP2A6 genotypes in individual susceptibility to environmentally induced diseases.

CHARLES UNIVERSITY Faculty of Pharmacy in Hradec Králové Department of Pharmacology & Toxicology Student: Jana Kahounová Supervizor: Doc. PharmDr. Marie Vopršálová, Ph.D. Ing. Jaroslav Procházka Title of diploma thesis: ELIMINATION OF ALKOHOL IN BLOOD OF PATIENTS TREATED WITH COUMARIN DRUGS Coumarin preparations are the most commonly used oral anticoagulants. The main representative is Warfarin. The most important function of warfarin is to reduce the formation of K-dependent coagulation factors in the liver. Alcohol is the oldest and most widely used addictive substance. Alcohol is the oldest and most commonly used substance. Society is tolerated and regular use of small doses of alcohol is considered "beneficial to health". The aim of my thesis was to find out how the concomitant use of coumarin drugs and alcohol interacts. There were tested 62 people (33 men and 29 women), of whom 27 people (14 men and 13 women) were treated with coumarin drugs, 35 people (19 men and 16 women) formed the control group of individuals without treatment with coumarin drugs. All people were given a standard dose of alcohol. The blood alcohol level (COBAS INTEGRA 400 plus) and INR (SYSMEX CA 1500) were measured in the time-0 (before administration), 1½ and 3 hours (after administration). The data obtained was...

Single-walled carbon nanotubes (SWNTs) are a new class of materials that have recently attracted a great deal of interest because of their unique structural, mechanical, and electronic properties. Also, SWNTs have a high potential for a number of technological applications, including molecular electronics, emissive devices, and photovoltaic devices. To fully utilize their unique properties, control of the solubility, processibility, and functionality of SWNTs is required. Therefore chemical functionalization of SWNTs using a variety of methods, in either covalent or noncovalent manner, has been developed to produce soluble nanotube composites coupled with various chemical moieties. To explore the possibility of making potential soluble nanotube-based materials for solar cells, SWNTs were functionalized with organic chromophore-labeled polymers via a radical coupling process. The organic chromophore was used to absorb light to produce photo-induced electrons, while the polymer chains were used for improving the solubility of SWNTs. These novel chromophore-labeled polymers were made by stable free radical polymerization (SFRP), either using a synthetic chromophore-functionalized styrenic monomer or by derivatizing well-defined polystyrenes. Specifically, the chromophores employed in this investigation were commercially available 7-hydroxycoumarin and coumarin-343. In order to carry out fluorescence studies of SWNT-coumarin composites systematically, various factors were probed by (1) altering polystyrene lengths between the SWNT and the coumarin; (2) changing the distribution of coumarins along the polymer chain, in the form of either a block or random copolymer; (3) placing single coumarins on the surface of SWNTs. All of these resulting polymer functionalized SWNTs were found to be soluble in certain organic solvents such as CHCl3. Different absorption behaviors have been observed for SWNTs functionalized with 7-hydroxycoumarin containing copolymers. Fluorescence was still observable for all of these composites, and the pi-pi interactions between coumarins and nanotubes were believed to be responsible for the broadening of emission bands of the resulting composites. / Thesis / Master of Science (MS)

Coumarin and cinnamate derivatives were positioned as either polymer chain ends or side groups to synthesize photoactive macromolecules and gain the ability to reversibly control molecular weight and crosslink density using UV light. The cinnamates and coumarins were reacted onto the polymers via multiple reaction pathways. Polymers were functionalized with coumarin or cinnamate groups via an esterification reaction between hydroxyl functionalities and an acid chloride derivatized coumarin group. In addition to the esterification reaction, cinnamates were also coupled to polymers via a ring opening reaction between a hydroxyl functionalized cinnamate derivative and a maleic anhydride repeat unit copolymerized into the polymer. Both functional groups undergo a [2π + 2π] photodimerization reaction (coumarin groups in the UVA and cinnamate groups in the UVB), which was utilized to crosslink and chain-extend macromolecules. Coumarin dimers possess the additional ability to photocleave and thus reverse when irradiated at 254 nm. The coumarin reversible photodimerization reaction was utilized to reversibly increase the molecular weight and molecular weight distribution of coumarin-functionalized PEG monols and diols. For example, the number average molecular weight of the coumarin-functionalized PEG diol doubled and the molecular weight distribution increased from 1.08 to 2.75 when exposed to 110 J cm⁻² of UVA irradiation. Subsequent photocleavage (UVC irradiation, 2 J cm⁻²) of the chain-extended PEGs, cleaved coumarin dimers decreasing the molecular weight and molecular weight distribution to their original values. A number of poly(alkyl acrylate) and poly(methyl acrylate) systems were functionalized with coumarin groups to study the effect of the glass transition temperature and alkyl ester side group composition on the photodimerization reaction and subsequent crosslinking. The glass transition temperature (T_g) acted as an on/off switch for the photodimerization reaction. While the absolute difference between T_g and irradiance temperature did not affect the rate or extent of photodimerization reaction, polymers with a T_g greater than the irradiance temperature displayed less reaction than those with a T_g lower than the irradiance temperature. The final extent of conversion was controlled by a complex combination of factors including alkyl ester side chain steric bulkiness. Coumarin-functionalized alkyl acrylates based on ethylhexyl acrylate were tested as detachable PSAs. A 98% decrease in the adhesive peel strength was observed after exposure to UVA irradiation. Cinnamate groups were utilized in the design and synthesis of UV-curable hot melt pressure sensitive adhesives (PSAs). The cinnamate groups were attached to the PSAs to provide a method to increase molecular weight and add a small amount of crosslinking leading to an increase the adhesive strength of the PSAs. Broadband UV irradiation from a laboratory scale industrial lamp increased the peel strength of the adhesives. Postcure of the irradiated cinnamate-functionalized UV-curable hot melt PSAs was reduced compared to photoinitiated free-radical photocurable UV-curable hot melt PSAs. / Ph. D.

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