Natural and synthetic heterocyclic cations that bind to the DNA minor groove have demonstrated effectiveness as therapeutic agents for cancer, parasitic and viral diseases, as well as powerful probes for use to extend our fundamental understanding of DNA molecular recognition. Crystal and NMR structures with a variety of minor groove binding compounds have shed light on the structural varieties of these systems, the important solvent molecules in the complexes, and the induced fit effects for binding of both DNA and the bound small molecule. Topics of specific importance in DNA recognition are the development of a greater variety of cell-permeable minor groove agents that have increased DNA binding sequence selectivity. In this dissertation, the structural and energetic basis of the interaction between DNA and minor groove binders has been systematically investigated. A set of powerful and complementary biophysical methods have been used: gel electrophoresis with ligation ladder assay, circular dichroism, mass spectrometry, surface plasmon resonance and isothermal titration calorimetry have been applied to determine the binding stoichiometry, binding affinity, kinetics and thermodynamics, and also the structural influence that minor groove binders can have on DNA. The results of several minor groove complexes clearly show that based on DNA sequences, minor groove binders can have multiple binding modes and consequently affect the geometry of DNA minor groove and the overall DNA curvature in distinct manners. In addition, the binding enthalpy of a minor groove binder is essentially salt concentration and binding mode independent. Besides the investigation of DNA-minor groove binder complex, the binding and inhibition of transcription factor PU.1 has also been studied. The highly positive charged PU.1 targets DNA by inserting an α-helix in the major groove of the 5’-GGAA-3’ site, and displays a strong salt concentration dependency. A set of minor groove binders have been rationally designed based on the high-affinity DNA sequence for PU.1 to target the flanking sequences of the 5’-GGAA-3’ site. They display a structure-related PU.1 inhibition efficacy. This work demonstrates that minor groove binders are capable of modulating PU.1 by targeting the opposite groove and supports future efforts to develop agents for other transcription factors.

Pin1 is an enzyme central to cell signaling pathways because it catalyzes the cis–trans isomerization of the peptide ω-bond in phosphorylated serine/threonine-proline motifs in many proteins. This regulatory function makes Pin1 a drug target in the treatment of various diseases. The effects of phosphorylation on Pin1 substrates and the basis for Pin1 recognition are not well understood. The conformational consequences of phosphorylation on Pin1 substrate analogues and the mechanism of recognition by the catalytic domain of Pin1 were determined using molecular dynamics simulations. Phosphorylation perturbs the backbone conformational space of Pin1 substrate analogues. It is also shown that Pin1 recognizes specific conformations of its substrate by conformational selection. Dynamical correlated motions in the free Pin1 enzyme are present in the enzyme of the enzyme–substrate complex when the substrate is in the transition state configuration. This suggests that these motions play a significant role during catalysis. These results provide a detailed mechanistic understanding of Pin1 substrate recognition that can be exploited for drug design purposes and further our understanding of the subtleties of post-translational phosphorylation and cis–trans isomerization. Results from accelerated molecular dynamics simulations indicate that catalysis occurs along a restricted path of the backbone configuration of the substrate, selecting specific subpopulations of the conformational space of the substrate in the active site of Pin1. The simulations show that the enzyme–substrate interactions are coupled to the state of the prolyl peptide bond during catalysis. The transition-state configuration of the substrate binds better than the cis and trans states to the catalytic domain of Pin1. This suggests that Pin1 catalyzes its substrate by noncovalently stabilizing the transition state. These results suggest an atomistic detail understanding of the catalytic mechanism of Pin1 that is necessary for the design of novel inhibitors and the treatment of several diseases. Additionally, a set of constant force biased molecular dynamics simulations are presented to explore the kinetic properties of a Pin1 substrate and its unphosphorylated analogue. The simulations indicate that the phosphorylated Pin1 substrate isomerizes slower than the unphosphorylated analogue. This is due to the lower diffusion constant for the phosphorylated Pin1 substrate.

is a functionally diverse large group of proteins sharing a jelly roll β-barrel fold. An enzymatic member 3-hydroxyanthranilate-3,4-dioxygenase (HAO) and a non-enzymatic member pirin, which is a human nuclear metalloprotein of unknown function present in all human tissues, were selected for structural and functional studies in this dissertation work. HAO is an important enzyme for tryptophan catabolism and for 2-nitrobenzoic acid biodegradation. In this work, seven catalytic intermediate were captured in HAO single crystals, enabling for the first time a nearly complete structural snapshot viewing of the entire molecular oxygen activation and insertion mechanism in an iron- and O2-depedent enzyme. The rapid catalytic turnover rate was found achieved in large part by protein dynamics that facilitates O2 binding to the catalytic iron, which is bound to the enzyme by a facile 2-His-1-carboxylate ligand motif. An iron storage and chaperon mechanism was also discovered in the bacterial source of this enzyme, which led to a proposed novel biological function of a mononuclear iron-sulfur center. Although human pirin protein shares the same structural fold with HAO, its iron ion is coordinated by a 3-His-1-carboxylate ligand motif. Pirin belongs to a subset of proteins whose members are playing regulatory functions in the superfamily. In this work, pirin is shown to act as a redox sensor for the NF-κB transcription factor, a critical mediator of intracellular signaling that has been linked to cellular responses to pro-inflammatory signals which controls the expression of a vast array of genes involved in immune and stress responses.

Cyclophilin A (CypA) is the well-studied member of a group of ubiquitous and evolutionarily conserved families of enzymes called peptidyl–prolyl isomerases (PPIases). These enzymes catalyze the cis-trans isomerization of peptidyl-prolyl bond in many proteins. The distinctive functional path triggered by each isomeric state of peptidyl-prolyl bond renders PPIase-catalyzed isomerization a molecular switching mechanism to be used on physiological demand. PPIase activity has been implicated in protein folding, signal transduction, and ion channel gating as well as pathological condition such as cancer, Alzheimer’s, and microbial infections. The more than five order of magnitude speed-up in the rate of peptidyl–prolyl cis–trans isomerization by CypA has been the target of intense research. Normal and accelerated molecular dynamic simulations were carried out to understand the catalytic mechanism of CypA in atomistic details. The results reaffirm transition state stabilization as the main factor in the astonishing enhancement in isomerization rate by enzyme. The ensuing intramolecular polarization, as a result of the loss of pseudo double bond character of the peptide bond at the transition state, was shown to contribute only about −1.0 kcal/mol to stabilizing the transition state. This relatively small contribution demonstrates that routinely used fixed charge classical force fields can reasonably describe these types of biological systems. The computational studies also revealed that the undemanding exchange of the free substrate between β- and α-helical regions is lost in the active site of the enzyme, where it is mainly in the β-region. The resultant relative change in conformational entropy favorably contributes to the free energy of stabilizing the transition state by CypA. The isomerization kinetics is strongly coupled to the enzyme motions while the chemical step and enzyme–substrate dynamics are in turn buckled to solvent fluctuations. The chemical step in the active site of the enzyme is therefore not separated from the fluctuations in the solvent. Of special interest is the nature of catalysis in a more realistic crowded environment, for example, the cell. Enzyme motions in such complicated medium are subjected to different viscosities and hydrodynamic properties, which could have implications for allosteric regulation and function.

Temporal and spatial molecular imaging of disease biomarkers using non-invasive MRI with high resolution is largely limited by lack of MRI contrast agents with high sensitivity, high specificity, optimized biodistribution and pharmacokinetics. In this dissertation, I report my Ph. D. work on the development of protein-based MRI contrast agents (ProCAs) specifically targeting different cancer biomarkers, such as grastrin-releasing peptide receptor (GRPR), prostate specific membrane antigen (PSMA), and vascular endothelial growth factor receptor-2 (VEGFR-2). Similar to non-targeted ProCAs, these biomarker-targeted ProCAs exhibit 5 - 10 times higher r1 and r2 relaxivites than that of clinical MRI contrast agents. In addition, these biomarker-targeted ProCAs have high Gd3+ binding affinities and metal selectivities. The highest binding affinity of the three GRPR-targeted contrast reagents obtained by grafting a GRPR ligand binding moiety into ProCA32 for GRPR is 2.7 x 10-9 M. We further demonstrate that GRPR-targeted ProCAs were able to semi-quantitatively evaluate GRPR expression levels in xenograft mice model by MRI. In addition, we have also created a PSMA-targeted ProCA which has a binding affinity to PSMA biomarker of 5.2 x 10-7 M. Further, we developed VEGFR-targeted contrast agent which is able to image VEGFR2 in mice models using T1-weighted and T2-weighted sequences. Moreover, the relaxivities and coordination water numbers of ProCAs can be tuned by protein design of ProCA4. Since disease biomarkers are expressed in various tumors and diseases, our results may have strong preclinical and clinical implications for the diagnosis and therapeutics of cancer and other type of diseases.

The development of novel selective 5-HT6 and 5-HT7 receptor antagonists is an ever-growing area of interest among medicinal chemists. The potential of developing a therapeutic agent useful as an antipsychotic or antidepressant, as well as the possibility to develop a drug for Alzheimer’s disease and obesity has led to an increase in synthesis of possible lead compounds. The synthesis of unfused biheteroaryl derivatives is described within. The derivatives have been evaluated for binding affinity at 5-HT2A, 5-HT6 and 5-HT7 receptors. The most potent 5-HT6 receptor antagonists include a benzene ring, a hydrophobic group and a protonated nitrogen atom. The most potent and selective compound synthesized is 1-[3-butyl-5-(thienyl)phenyl]-4-methylpiperazine. The binding site of the 5-HT7 receptor is similar to that of the 5-HT6 receptor and the most selective and potent 5-HT7 receptor antagonist also contains a potonated nitrogen atom and a hydrophobic group. The difference in selectivity between the 5-HT6 and 5-HT7 receptor antagonists is the aromatic ring. The most potent 5-HT7 receptor antagonist synthesized contains a pyridine ring instead of benzene, as in the 5-HT6 receptor antagonist. The most potent and selective 5-HT7 receptor antagonist is 1-[4-(3-furyl)-6-methylpyridin-2-yl]-4-methylpiperazine. The need to increase selectivity for both 5-HT6 and 5-HT7 receptors has led to the synthesis of flexible-chain linked derivatives and the results are described within.

The development of new reagents to efficiently cleave peptides and proteins has become increasingly important for protein structural studies and other applications. However, this has proved to be a very challenging task due to the extreme stability of the peptide amide bond. Transition metal complexes cleave proteins and peptides through either oxidative or hydrolytic pathways. However, hydrolytic cleavage is preferred over oxidative cleavage, because the latter process produces irreversibly modified peptide fragments. Metal-assisted peptide hydrolysis is introduced in Chapter I. The metals Ce(IV), Co(II), Co(III), Cu(II), Fe(III), Mo(IV), Ni(II), Pd(II), Pt(II), Zn(II), and Zr(IV) are described as promising non-enzymatic hydrolysis reagents. In Chapter II, Zr(IV)-assisted hydrolysis of the dipeptide Gly-Gly and of its N- and C- blocked analogs is described. The highest levels of cleavage were observed at pH values ranging from 4.4 to 4.7. When the pH was raised to ~ 7.0, hydrolysis yields were decreased and amounts of zirconium precipitation were increased proportionately. Zirconium(IV)-assisted peptide hydrolysis in the presence of 4,13-diaza-18-crown-6 is reported in Chapter III. The goal of this work was to use an azacrown ether to reduce Zr(IV) precipitation and enhance levels of hydrolysis at neutral pH. An experiment in which 16 glycine containing dipeptides were hydrolyzed by Zr(IV) and by Zr(IV)/4,13-diaza-18-crown-6 indicated that 4,13-diaza-18-crown-6 markedly enhanced the reactivity of Zr(IV) under near physiological conditions. Because Zr(IV) precipitation was not reduced in these reactions, we proposed that hydrolysis of peptides by Zr(IV)/4,13-diaza-18-crown-6 might be heterogeneous in nature. In Chapter IV, seventeen macrocyclic and open-chain Zr(IV) ligands were compared in order to gain mechanistic insights that would enable hydrolysis yields at neutral pH to be further improved. While the macrocyclic ligands 4,13-diaza-18-crown-6 and 4,10-trioxa-7,13-diazacyclopentadecane tended to produce higher levels of Zr(IV)-assisted dipeptide cleavage, it was not necessary to have a ring structure to enhance Zr(IV) reactivity. With respect to the open-chain ligands, the potential ability to form multiple chelate rings appeared to coincide with reduced levels of Zr(IV) precipitation as well as with reduced levels of dipeptide hydrolysis. In Chapter V, a summary of our results and conclusions is presented.

This dissertation describes the advantages and feasibility of newly synthesized near-infrared (NIR) bis-heptamethine cyanine (BHmC) dyes for non-covalent labeling schemes. The NIR BHmCs were synthesized for biomolecule assay. The advantages of NIR BHmCs for biomolecule labeling and the instrumental advantages of the near-infrared region are also demonstrated. Chapter 1 introduces the theory and applications of dye chemistry. For bioanalysis, this chapter presents covalent and non-covalent labeling. The covalent labeling depends on the functionality of amino acids and the non-covalent labeling relies on the binding site of a protein. Due to the complicated binding process in non-covalent labeling, this chapter also discusses the binding equilibria in spectroscopic and chromatographic analyses. Chapter 2 and 3 evaluate the novel BHmCs for non-covalent labeling with human serum albumin (HSA) and report the influence of micro-environment on BHmCs. The interesting character of BHmCs in aqueous solutions is that the dyes exhibit non- or low-fluorescence compared to their monomer counterpart, RK780. It is due to their H-type closed clam-shell form in the solutions. The addition of HSA or organic solvents opens up the clam-shell form and enhances fluorescence. The binding equilibria are also examed. Chapter 4 provides a brief introduction that summaries the use of capillary electrophoresis (CE), and offers a detailed instrumentation that discusses the importance and advantage of a detector in NIR region for CE separation. Chapter 5 focuses on the use of NIR cyanine dyes with capillary electrcophoresis with near-infrared laser induce fluorescence (CE-NIR-LIF) detection. The NIR dyes with different functional groups show that RK780 is a suitable NIR dye for HSA labeling. The use of BHmCs with CE-NIR-LIF reduces signal noises that are commonly caused by the interaction between NIR cyanine dyes and negatively charged capillary wall. In addition, bovine carbonic anhydrase II (BCA II) is applied to study the influence of hydrophobicity on non-covalent labeling. Finally, chapter 6 presents the conformational dependency of BHmCs on the mobility in capillary and evaluates the further possibility of BHmCs for small molecule detection. Acridine orange (AO) is used as a sample and it breaks up the aggregate and enhances fluorescence. The inserted AO into BHmC changes the mobility in capillary, owing to the conformational changes by AO.

Nucleic acids are responsible for the storage of genetic information and directly participate in gene replication, transcription and expression, and thereby the control of nucleic acids leads to the regulation of genetic information flow and gene expression. Meanwhile, many non-coding RNAs are in-volved in signal transduction directly. Moreover, nucleic acid-based therapeutic strategies have been lead to drug candidates and are effective tools in drug discovery and disease study at the molecular level as well as the genetic level. Consequently, the 3D crystal structure study and related functional research on natural and unnatural nucleic acids have become very popular area, expanding their potential appli-cation in medicinal and biological chemistry. Since oxygen, sulfur, selenium and tellurium are in the same elemental family (VIA) in the peri-odic table, we anticipate that oxygen atoms in nucleic acids can most likely be replaced with the other chalcogen atoms without causing significant perturbations. Owing to the special K edge and unique properties of selenium, our lab has completed the chemical and enzymatic synthesis of unnatural nucle- ic acids with selenium substitutions at various positions. The selenium functionality in nucleic acid is es-sential for nucleic acids’ structural determination at the atomic level. Additionally this novel elemental feature (atomic size and electronic nature) provides nucleic acids with unique properties. In addition, the selenium derivatization can facilitate crystal growth. Other chalcogen elements are applicable as well to modify nucleic acid, generating some special biofunctions, like the application of phosphorthioate oligonucleotide in gene therapy. This dissertation will outline the chalcogen elements (especially selenium) modifications of nucleic acids, including syntheses strategies, structure studies and potential therapeutic applications. Our research work here tries to show that (1) Selenium functionality is able to facilitate the crystal structure determination, by both helping solve phase problem and accel-erating crystal growth; (2) Selenium functionality can generate special capability to nucleic acids, like improved base pair fidelity, novel atomic interactions and feasibility to be biological chemistry probe; (3) Selenium derivatized oligonucleotides are extraordinary good candidates for gene therapy discovery, considering its stability under nuclease environment. In general, these atom-specific replacements gen-erate a new paradigm of nucleic acids. INDEX WORDS: Nucleic acid, Selenium, X-ray crystal structure, Biofunction, Therapeutics

Fibrinogen aggregation is the last step in blood coagulation. Inhibition of fibrinogen aggregation could lead to anticoagulation effects. However, there is no good method for the ready evaluation of fibrinogen coagulation. A commonly used path method is slow and requires an expensive instrument. In this project, we have developed a microplate reader and In evaluating inhibitors of fibrinogen coagulations there is no good method. As an important process in hemostasis, fibrinogen coagulation is often detected by micro-plate reader. In our test of fibrinogen coagulation, we improved the observing and analyzing method by using photograph to see concentration-depended effect of thrombin inhibitors on the coagulation. Three known thrombin inhibitors, AEBSF, APMSF and PMSF, were applied to develop the method for detecting the fibrinogen coagulation. The results showed our method is of accuracy in determination of the amount of fibrin when compared with other types of methods.