In the HIV viral integration procedure, 3’-processing of the viral DNA by the integrase enzyme is an essential first step which is followed by the integration of viral DNA into the host genome. In 3’-processing, the integrase cleaves the backbone of the DNA substrate on the 3’ end of a conserved CA dinucleotide motif and inserts a helix between the two DNA strands, forcing them apart (Hare, S., 2012). Our study confirms that the presence of a G-amino group is crucial for 3’-processing. Substituting inosine for G in the CA step removes this amino group and results in loss of enzyme activity. Further work showed that the presence of a terminal duplex segment is not required for 3’-processing. Additional substrate modifications are studied in order to evaluate the actual importance of the CA step.

The most critical biochemistry in an organism supports the central dogma of molecular biology: transcription of DNA to RNA and translation of RNA to peptide sequence. Proteins are then responsible for catalyzing, regulating and ensuring the fidelity of transcription and translation. At the heart of these processes lie selective biomolecular interactions and specific dynamics that are necessary for complex formation and catalytic activity. Through advanced biophysical and computational methods, it has become possible to probe these macromolecular dynamics and interactions at the molecular and atomic levels to tease out their underlying physical bases. To the end of a more thorough understanding of these physical bases, we have performed studies to probe the motions and interactions intrinsic to the function of biomolecular complexes: modeling the dual-base flipping strategy of alkylpurine glycosylase D, dynamically tracing evolution and epistasis in the 3-ketosteroid family of nuclear receptors, discovering the allosteric and conformational aspects of transcription regulation in liver receptor homologue 1, leveraging specific contacts in tyrosyl-DNA phosphodiesterase 2 for the development of novel inhibitor scaffolds, and detailing the experimentally observed connection between solvation and sequence-specific binding affinity in PU.1-DNA complexes at the atomic level. While each study seeks to solve system-specific problems, the collection outlines a general and broadly applicable description of the biophysical motivations of biochemical processes.

In this paper, an improved method [1] for the chemical synthesis of 2SeU-RNA was reported using a streamlined strategy employs 2'-O-Thiomopholine-4-carbothioate protecting group. And single step deprotection of the resulting oligoribonucleotide product using 1,2-diamines/toluene under anhydrous conditions would retain the Selenium atom introduced on the 2-possiton of the modified Uracil. The process is doable with most standard heterobase protection and deprotection, it greatly simplifies the synthesis of 2SeU-RNAs and can be applied to other Selenium modified RNAs synthesis. It makes the synthesis of RNA become as simple and efficient as the chemical synthesis of DNA. Furthermore, the design and synthesis of self-assembling 2SeU-RNA square are reported which enable further structure studies and application of unique 2SeU-RNAs.

Choline oxidase catalyzes the oxidation of choline to glycine betaine. The reaction includes betaine aldehyde as an intermediate. FAD is reduced by the alcohol substrate, betaine aldehyde intermediate and oxidized by molecular oxygen to give hydrogen peroxide. In this study, the Ser101Ala variant of choline oxidase was prepared to elucidate the contribution of the hydroxyl group of Ser101 in the proton and hydride transfer reactions for proper preorganization and reorganization of the active site towards quantum mechanical tunneling. The thermodynamic parameters associated with the enzyme-catalyzed OH and CH bond cleavages and the temperature dependence of the associated solvent and substrate kinetic isotope effects were investigated using a stopped-flow spectrophotometer. The proton and hydride transfer have been shown to be occurring via quantum tunneling in CHO-S101A enzyme.

Development of a simple, specific, sensitive and rapid RNA microchip for detection of Head and Neck Cancer (HNC) mRNA, pathogenic bacteria and dengue virus (DENV) RNA is reported. By use of nucleases and polymerases specific RNAs are selectively labeled and detected without separation, reverse transcription and or polymerase chain reaction. This is accomplished by designing specific Hybrid probes consisting of DNA-2’-O-Me-RNA-DNA regions to target the RNA of interest. Upon hybridization with the target RNA, RNase H digestion is used to remove the 3’- RNA sequences which exposes the template for Klenow extension with reporter molecules such as hapten or fluorophore labels. This novel RNA microchip is fast (ca. 1 h detection time), selective as individual RNAs are detected in a synthetic mixture and total RNA mixtures, specific for single nucleotide polymorphisms (SNPs) discrimination and sensitive up to attomole level for chemiluminescence detection and lower femtomole for gold nanoparticles (AuNPs) and silver staining method. Using chemiluminescence, HNC biomarkers, VCAM1 and IL8 are specifically labeled and detected in the presence of thousands of other mRNAs in cancer cell lines and human colon cancer total RNA without interference. Furthermore, the method is highly specific as shown with DENV SNPs discrimination. Moreover, we report rapid (ca 1hour), selective, specific multi-marker detection of pathogenic mRNAs and HNC mRNAs using AuNPs-silver staining on the RNA microchip. Streptavidin gold nanoparticles technology has a potential in the analysis of specific mRNAs in a wide array of field including infectious diseases diagnosis, viral infections, food safety, gene expression profiling and cancer detection. A simple and rapid NaOH RNA extraction procedure was developed for E. coli total RNA extraction with specific results on the RNA microchip using both chemiluminescence and AuNPs silver staining. This extraction avoids the use of commercial RNA purification kits thus reducing the cost. Furthermore, visual detection on the RNA microchip is simple, does not require electricity or special equipment, and therefore is a good candidate for field diagnostics with minimum resources.

Carbocyanine dyes are organic compounds containing chains of conjugated methine groups with electron-donating and electron-withdrawing substituents at the terminal heterocycles of the general formula [R1-(CH)n-R2]+X-. The synthetic methodology and optical properties of carbocyanines will be discussed. This thesis consists of two parts: (A) synthesis and optical properties of novel carbocyanine dyes substituted with various amines and the synthesis of unsymmetrical carbocyanine dyes containing monofunctional groups for bioconjugation. (B) synthesis of heptamethine carbocyanine dyes to be used for image-guided surgery. In part A, the synthesis of carbocyanine dyes functionalized with various amines and studies of their optical properties with respect to absorbance, fluorescence, quantum yield and extinction coefficient will be presented. These property studies will aid in designing efficient dyes for future biomedical applications. Part A will also include a one pot synthesis of unsymmetrical carbocyanine dyes functionalized with mono carboxylic acid chains, useful for biomolecule (i.e. proteins, amino acids, etc.) conjugation. Part B will describe the synthesis of novel carbocyanine dyes to be used for cancer image-guided surgery. Cancers are thus far incurable diseases, i.e. there are no drugs currently available to cure cancer; however, by designing a dye to visualize tumor cells will greatly increase the efficiency of cancer removal and hopefully increase the survival rate of cancer patients. The dyes reported in this thesis are superior to commercially available dyes used to visualize and identify various tumors invisible to the naked eye of surgeons with regards to biodistribution and clearance through kidney filtration.

3-Chloro-4,4,5-trimethyl-3,5-diphenyl-4,5-dihydro-3H-pyrazole (3b) and 3-bromo-4,4,5-trimethyl-3,5-diphenyl-4,5-dihydro-3H-pyrazole (3c) were prepared for the thermolysis project. The thermal decompositions of 3b and 3c were monitored using 1H NMR spectroscopy. Plots of ln (% starting material) vs. time (sec) were linear for at least two half lives and the first order rate constants were determined over at least a 30o temperature range. The relative reactivity was found to be 3c > 3b. The activation parameters determined for the thermal decomposition of the pyrazoline at 150oC were found to be: for 3b &#;H‡ = 33 &#;1.0 kcal/mol, &#;S‡ = -2.4 &#; 0.07eu , k150 0 = 7.34 &#; 0.44 x 10 -5 s-1 ; for 3c &#;H‡ = 30&#;0.2 kcal/mol, &#;S‡ = -6.9 &#;0.03 eu, k150o = 42.3&#;0.7 x 10-5 s-1. Thermal decomposition of 3b both neat and in dibromobenzene (DBB) resulted in the formation of an intermediate 2,3-diphenyl-4-methyl-1,3-pentadiene (8) as a major product and minor isomers of 8. These intermediates then thermally decomposed to 1,1,3-trimethyl-2-phenyl-1H-indene (9) via an acid catalyzed process. In order to gain a mechanistic understanding (ionic vs. radical pathways) of the thermal decomposition of 3b, a product study was conducted in protic solvents. In methanol and ethanol, 3b underwent an ionic reaction (SN1-type) with the solvent to produce 3-methoxy/ethoxy-4,4,5-trimethyl-3,5-diphenyl-4,5-dihydro-3H-pyrazole (3/3d) in good yield. The reaction of 3b with refluxing protic solvents led to the development of new method for the synthesis of alkoxy-4,5-dihydro-3H-pyrazoles which is both safe and efficient.

A series of penta-aryl/alkyl-1-(toluene-4-sulfonyl)-4,5-dihydro-1H-pyrazole 5a-c was synthesized by addition of methyllithium or phenylllithium followed by trapping the nitrogen anion intermediate with tosyl-fluoride to cyclic azines 2a,b. Addition of methyllithium or phenyllithium to 5a-c generated a series of hexa-aryl/alkylsubstituted-4,5-dihydro-3H-pyrazoles 6a-c. Neat thermolysis of hexa-aryl/alkylsubstituted-4,5-dihydro-3H-pyrazoles 6a-c at 200◦C produced hexa-aryl/alkylsubstituted cyclopropanes 7a-c in high yield.

Dicationic diamidines synthesized in the Boykin group a have shown broad range of activity against a wide variety of microbial pathogens such as Cryptosporidium parvum, Leishmania donovani, Plasmodium falciparum, Trypanosoma burcei, and Trichomonas vaginalis. Thiophene based dicationic diamidines have been especially impressive versus Trichomoniasis, and several species of the leishmania parasite. The best compound in vitro against leishmania was 2,5-bis-(4-amidophenyl)thiophene [DB 351]. In an attempt to improve upon antileishmanial activity, several analogs of DB 351 have been synthesized. Previously the central heterocyclic ring has been changed (furan, pyrrole, etc.), and the phenyl has been substituted with a pyridine ring, however, in the thiophene series, the 3 and 4 position of the central thiophene ring has remained unmodified. By modifying the 3 and 4 position of the thiophene ring, and taking advantage of the substituent effect, the pharmacodynamic and distribution properties of DB 351 will altered; hopefully leading to more potent antileishmanial compounds.

Choline oxidase from Arthrobacter globiformis is a flavin-dependent enzyme that catalyzes the oxidation of choline to betaine aldehyde through two sequential hydride-transfer steps. The study of this enzyme is of importance to the understanding of glycine betaine biosynthesis found in pathogenic bacterial or economic relevant crop plants as a response to temperature and salt stress in adverse environment. In this study, chemical modification of choline oxidase using two irreversible inhibitors, tetranitromethane and phenylhydrazine, was performed in order to gain insights into the active site structure of the enzyme. Choline oxidase can also be inactivated irreversibly by freezing in 20 mM sodium phosphate and 20 mM sodium pyrophosphate at pH 6 and -20 oC. The results showed that enzyme inactivation was due to a localized conformational change associated with the ionization of a group in close proximity to the flavin cofactor and led to a complete lost of catalytic activity.