Small molecule mediated chemical intervention of biological processes using nucleic acid targets has proven extremely successful and is continually providing exciting new avenues for the development of anti-cancer agents and molecular probes. Among the alternative DNA confrormations, G-quadruplexes has certainly garnered much recognition due to increase in evidences supporting their involvement in diverse biological process. The grooves of the quadruplexes offer an alternate recognition site for ligand interactions with potentially higher selectivity than the traditional terminal stacking sites. DB832, a bifuryl-phenyl diamidine, was recently reported to selectively recognize human telomeric G-quadruplex, as a stacked species, with significant selectivity over duplex sequences. A series of biophysical studies were conducted to test the groove-binding mode of DB832, along with the selectivity for diverse quadruplex forming sequences. To gain better understanding of quadruplex groove-recognition by DB832, a series of structurally similar heterocyclic diamidines were also evaluated. The unique binding mode of DB832 may allow it to serve as a paradigm for the design of new class of highly selective quadruplex groove-binding molecules. Beyond the alternative secondary structures, it is also becoming increasingly apparent that the structure and dynamics of the canonical Watson–Crick DNA double helix play pivotal roles in diverse biological functions. DB1878, a phenyl-furan-indole diamidine, was shown to recognize a mixed GC/AT motif as a stacked antiparallel dimer, and a detailed structural analysis is reported here. Interestingly, the DNA recognition is completely different from the reported molecules in literature, and represents an entirely new motif for DNA minor groove recognition.

Histone acetyltransferases (HATs) are important enzymes in transcriptional control and potential targets for chemotherapeutic intervention in malignant diseases. Among different HAT members, the yeast Esa1 and human Tip60 (the HIV-1 Tat interactive protein, 60KDa) play multiple roles in normal cellular processes including transcription, cell cycle and checkpoint machinery, double strand DNA break repair, apoptosis, and cell cycle progression. Tip60 is also implicated in several human diseases such as prostate cancer, and gastric cancer. These studies suggest that Tip60 is a potential therapeutic target for new cancer treatment. So, we designed experimental work to synthesize and investigate organic inhibitors of Tip60 using different strategies, including substrate analogs, small molecule screening, and modification of the natural product anacardic acid. These studies provide important chemical agents for basic biology research of HAT function, and produce potential lead compounds for future pharmacologic intervention of HAT deregulation in cancer. Currently, of the methods used for the measurement of acetyltransferase activities, many comprise tedious separation procedures and involve enzyme-coupled steps or radioactive materials. These shortcomings have limited their applications in high-throughput screening (HTS) of HAT inhibitors. To circumvent these problems, a homogenous fluorescent HAT assay based on engineered H4 peptide was designed, synthesized, and evaluated. The data showed that these fluorescent reporters can be used to detect the acetyltransferase activities.

Essential metals like Ca2+ and Zn2+ play critical roles in biological processes through protein interactions. Conversely, non-essential metals (e.g., Gd3+ and Pb2+) also interact with proteins, often with toxic effects. Molecular metal toxicity is assumed to be due to ionic displacement, and studies have demonstrated that Pb2+ replaces Zn2+, Ca2+ and other essential metals in proteins. The focus of this work was to compare protein Ca2+ and Pb2+ -binding sites and to investigate a mechanism of Pb2+ toxicity in Ca2+-binding proteins, particularly the intracellular trigger protein calmodulin (CaM) which binds four Ca2+ ions and interacts with numerous molecular targets via Ca2+-induced conformational change. A statistical analysis of PDB structural data for Pb2+ and Ca2+-binding (EF-hand and non-EF-hand) proteins revealed fewer binding ligands in Pb2+ sites (4 ± 2), than non-EF-Hand (6 ± 2) and EF-Hand (7 ± 1) Ca2+-binding sites. Pb2+ binds predominantly with sidechain Glu (38.4%), which is less prevalent in both non-EF-Hand (10.4%) and EF-Hand (26.6%) sites. Interestingly, analyses of proteins where Pb2+ replaces Ca2+ (calmodulin) or Zn2+ (5-aminolaevulinic acid dehydratase) revealed structural changes presumably unrelated to ionic displacement. These results suggested that Pb2+ adopts diverse binding geometries and that opportunistic binding outside of known Ca2+-binding sites may play a role in molecular metal toxicity. Ca2+-binding affinities (Kd) using phenylalanine and tyrosine fluorescence were found to be 1.15 ± 0.68 X 10-5 M and 2.04 ± 0.02 X 10-6 M for the N- and C-terminal domains, respectively. The Kd for Pb2+-binding in the N-terminal domain, 1.40 ± 0.30 X 10-6 M, was 8-fold higher than Ca2+. Binding of Pb2+ in the C-terminal domain produced a biphasic response with Kd values 7.34 ± 0.95 X 10-7 M and 1.93 ± 0.32 X 10-6 M, suggesting a single higher affinity Pb2+-binding site in the C-terminal domain with nearly equivalent affinity for the remaining sites. Competitive effects of Pb2+ added to Ca2+-loaded CaM were examined using multiple NMR techniques. Pb2+ was found to displace Ca2+ only in the N-terminal domain, however structural/dynamic changes were observed in the central helix apparently due to Pb2+-binding in secondary sites. These data supported our hypothesis that CaM structure and function is altered by opportunistic Pb2+-binding.

DNA polymerase binds to the double stranded DNA and extends the primer strand by adding deoxyribonucletide to the 3’-end. Several reactions in the polymerase active site have been reported by Kornberg in addition to the polymerization. We observed DNA polymerase I can act as a pyrophosphatase and hydrolyze deoxyribonucletide. In performing the pyrophosphatase activity, DNA polymerase I requires to interact with RNA. RNA in general, was found to activate the DNA polymerase I as pyrophosphatase. This hydrolysis causes depletion of dNTP and inhibits DNA polymeration synthesis in vitro. In this RNA-dependent catalysis, DNA polymerase I catalyzes only dNTP but not rNTP. We have also observed that many other DNA polymerases have this type of the RNA-dependent pyrophosphatase activity. Our experimental data suggest that the exonuclease active sites most likely play the critical role in this RNA-dependent dNTP hydrolysis, which might have a broader impact on biological systems. On the basis of the crystal structure of a ternary complex of RNase H (Bacillus halodurans), DNA, and RNA, we have introduced the selenium modification at the 6-position of guanine (G) by replacing the oxygen (SeG). The SeG has been incorporated into DNA (6 nt. - 6 nucleotides) by solid phase synthesis. The crystal structure and biochemical studies with the modified SeG-DNA indicate that the SeDNA can base-pair with the RNA substrate and serve as a template for the RNA hydrolysis. In the crystal structure, it has been observed that the selenium introduction causes shifting (or unwinding) of the G-C base pair by 0.3 Å. Furthermore, the Se-modification can significately enhance the phosphate backbone cleavage (over 1000 fold) of the RNA substrate, although the modifications are remotely located on the DNA bases. This enhancement in the catalytic step is probably attributed to the unwinding of the local duplex, which shifts scissile phosphate bond towards the enzyme active site. Our structural, kinetic and thermodynamic investigations suggest a novel mechanism of RNase H catalysis, which was revealed by the atom-specific selenium modification.

Choline oxidase catalyzes the flavin-dependent, two-step oxidation of choline to glycine betaine via the formation of an aldehyde intermediate. The oxidation of choline includes two reductive half-reactions followed by oxidative half-reactions. In the first oxidation reaction, the alcohol substrate is activated to its alkoxide via proton abstraction and oxidized via transfer of a hydride from the alkoxide α-carbon to the N(5) atom of the enzyme-bound flavin. In the wild-type enzyme, proton and hydride transfers are mechanistically and kinetically uncoupled. The role of Ser101 was investigated in this dissertation. Replacement of Ser101 with threonine, alanine, cysteine, or valine demonstrated the importance of the hydroxyl group of Ser101 in proton abstraction and in hydride transfer. Moreover, the kinetic studies on the Ser101Ala variant have revealed the importance of a specific residue for the optimization of the overall turnover of choline oxidase. The UV-visbible absorbance of Ser101Cys suggests Cys101 can form an adduct with the C4a atom of the flavin. The mechanism of formation of the C4a-cysteinyl adduct has been elucidated. D-arginine dehydrogenase (DADH) catalyzes the oxidation of D-amino acids to the corresponding imino acids, which are non-enzymatically hydrolyzed to α-keto acids and ammonia. The enzyme is strick dehrogenase and deoesnot react with molecular oxygen. Steady state kinetic studies wirh D-arginine and D-histidine as a substrate and PMS as the electron acceptor has been investigated. The enzyme has broad substrate specificity for D-amino acids except aspartate, glutamate and glycine, with preference for arginine and lysine. Leucine is the slowest substrate in which steady state kinetic parameters can be obtained. The chemical mechanism of leucine dehydrogenation catalyzed by DADH was explored with a combination of pH, substrate and solvent kinetic isotope effects (KIE) and proton inventories by using rapid kinetics in a stopped-flow spectrophotometer. The data are discussed in the context of the crystallographic structures at high resolutions (<1.3 Å) of the enzyme in complex with iminoarginine or iminohistidine.

Calmodulin (CaM) is a ubiquitous intracellular protein that regulates biological activities of numerous enzymes and ion channels. Upon responding Ca2+ concentration change, Ca2+- dependent CaM activates the hydrolyzation of cGMP by PDE and Ca2+ releasing channel activity of ryanodine receptor. In this dissertation, a series of CaM variants were engineered to enhance Ca2+ binding affinities by increasing the number of negative charged residues in individual EF-hand. The capability of shifting the biphasic Ca2+-activation profile of RyR1 is significantly altered by changing Ca2+ binding affinity of CaM at the C-terminal. This indicates that examining Ca2+-CaM affinity is a valid strategy to tune the activation profile of CaM-regulated ion channels. To further understand interactions between CaM and RyR1, NMR was used to determine their binding mode. To dissect roles of structural components of CaM in metal binding and regulation of biological functions of target proteins, we created half-CaMs and Del-CaM. Binding affinities of these variants to Ca2+, Tb3+ and Gd3+ were determined by fluorescence spectroscopy; functional studies were conducted using single channel analysis and PDE function assay. Another objective of my dissertation is to design a protein based contrast agent for molecular imaging. CaM was selected as the scaffold protein for designing Gd3+ based MRI contrast agent by modifying metal binding sites as well as grafting a biomarker peptide into the linker region to specifically target cancers with efficient and optimized modifications. The physical kinetic properties and animal imaging effects of these designed contrast agents were investigated by various methods.

The previous view of DNA as a linear sequence of bases is evolving to consider structure, topology and dynamics. Sequences surrounding damage lesions have been shown to effect enzyme recognition and processing. Here we present an in depth investigation of subtle structural and dynamical features imparted to nucleic acid duplexes by a designed modification or damage lesions. Highly restrained solution structures were generated and validated utilizing a range of NMR techniques. This allowed for the characterization of multiple features of the nucleic acid duplex; such as base pairing, backbone torsion angles, deoxyribose sugar pucker, and intra and inter nucleotide proton distances. Additional experiments provided insight into dynamic movements of the nucleic bases. These features are then correlated to enzyme data in order to explain the observed modulation of activity.

Carbohydrates are known to play important roles in a large number of physiological and pathological processes. Conceivably, “binders” of carbohydrates of biological importance could be used as diagnostic and therapeutic agents. Currently, lectins are the major available tools in research for carbohydrate recognition. However, the available lectins often have cross-reactivity issues, along with the high costs and stability issues. Therefore, there is a critical need to develop alternatives (lectin mimics). In this regard, there have been very active efforts in developing different “binders”, such as small molecule lectinmimics and aptamers. Among all the small molecule lectinbmimics developments, boronic acid stands out as the most important building blocks of the sensors design for carbohydrates biomarkers due to its intrinsic binding affinities with diols. To address a fundamental question that whether boronic acid also binds to six-membered ring sugars, with very limited precedents, we provided a concrete experimental evidence of the binding. Specifically, a series of isoquinolinylboronic acids were found to have remarkably high binding affinities with fluorescence change upon binding to representative sugars. Most importantly, these isoquinolinylboronic aicds showed weak but very encouraging bindings with six-membered sugar model. All these promising results paves the way of using boronic acids, especially isoquinolinylboronic acid as building blocks for chemosensors design for biological carbohydrates biomarkers, which universally contain six-membered ring and liner diols. Aptamer provides another alternative way for sensors development for carbohydrates biomarkers as lectin mimics. Compared to lectins, they are normally cheaper and more stable. However, there is much less options. Another challenging area for aptamer-based lectin mimics development is the difficulty to differentiate changes in glycosylation patterns of a glycoprotein, which affect the function of a glycoprotein and thus recognized as biomarkers. To address this major challenge, our group first demonstrated that the incorporation of a boronic acid into DNA would allow for the aptamer selection process to gravitate towards the glycosylation site. To examine the generality of boronic acid incorporation, increase the structural diversity, and broaden the application of boronic acid-modified DNA, a series of B-TTP analogues with simplified structures were designed, synthesized, and successfully incorporated into DNA. A simple route was also developed using 1,7-octadiyne as a linker for both Sonogashira coupling with thymidine and CuAAC tethering of a boronic acid moiety. This paves the way for the preparation of a large number of B-TTPs with different structural features for aptamer selection or array analysis. Finally, bacterial quorum sensing has received much attention in recent years because of its relevance to pathological events such as biofilm formation. As one of the very first groups that developed a series of antagonists for AI-2 mediated quorum sensing, we herein designed and synthesized a series of analogues based on the structures of two lead inhibitors identified through virtual screening. Besides, we also examined their inhibitory activities, twelve of which showed equal or better inhibitory activities compared with the lead inhibitors. The best compound showed an IC50 of about 6 mM in a whole cell assay using Vibrio harveyi as the model organism. This encouraging results and SAR discuss also paves the way for the finding of more potent compound through further structure optimization.

Heptamethine cyanine dyes are attractive compounds for imaging purposes in biomedical applications because of their chemical and photophysical properties exhibited in the near-infrared region. A series of meso amino-substituted heptamethine cyanine dyes with indolenine, benz[e]indolenine and benz[c,d]indolenine heterocyclic moieties were synthesized and their spectral properties including fluorescence quntum yield were investigated in ethanol and ethanol/water mixture. Upon substitution with amines, the absorption maxima of the dyes shifted to the lower wavelength region (~600 nm), showed larger Stokes shifts and stronger fluorescence which can be attributed to an excited state intramolecular charge transfer (ICT). High quantum yields were observed for primary amine derivatives and lower quantum yields were observed for secondary amine derivatives. Fluorescence quantum yields are greater for dyes with 3H-indolenine terminal moieties than for dyes with benz[e]indolenine end groups. Benz[c,d]indolenine based heptamethine cyanine dyes exhibited the lowest quantum yield due to aggregation in solution. In general, the benz[e]indolenine hepatemethine cyanines showed high Stokes shifts compared to indolenine dyes. For the meso-chloro dyes, the absorption maxima for the dyes shifted bathochromically in the order of indolenine, benz[e]indolenine and benz[c,d]indolenine.

Micellar electrokinetic chromatography (MEKC) and capillary electrochromatography (CEC) are two of the major capillary electrophoresis (CE) modes that have been interfaced to mass spectrometry (MS) for sensitive and selective analysis of chiral compounds. This research combines these two modes and expands their applications in chiral CE analysis. Chapter 1 is a review of amino acid based molecular micelles used in MEKC-MS for enantioselective analysis over the past five years. In this chapter, a typical MEKC-MS experiment setup as well as detailed standard operating procedure in synthesis of molecular micelles and running a typical MEKC-MS experiment using the molecular micelles is discussed. Chapter 2 described a multivariate MEKC-MS optimization for the simultaneous analysis of two negatively charged model chiral compounds in negative ion mode with molecular micelles. In this chapter, a central composite design (CCD) is used to first construct a series of experiments to optimize all the important MEKC-MS parameters. Next, response surface methodology (RSM) was used to analyze the interactions between the factors, picking up the best separation and detection conditions, predicting the result of the chiral separation/MS detection, and finally running the actual experiment and comparing the chromatographic results with the predicted parameters. Chapter 3 demonstrates a similar multivariate MEKC-MS optimization for analysis of a positively charged model chiral compound in a positive ion mode. The same CCD and RSM methods were used to optimize the separations and MS sensitivity. Chapter 4 describes a chiral analysis of four neutral benzoin derivatives (hydrobenzoin, benzoin, benzoin methyl ether, and benzoin ethyl ether) using MEKC coupled to atmospheric pressure photo-ionization mass spectrometry (APPI-MS). The same multivariate experimental design strategy was used to optimize the MEKC as well as APPI-MS parameters. Simultaneous chiral separation of all four benzoin derivatives was achieved with high detection sensitivity compared to UV-detection. Chapter 5 introduces a novel one-pot synthesis scheme for an acryloyl-terminated, carbamate-linked surfactant-bound monolith with leucine head group and different chain lengths. The method promises to open up the discovery of new amino acid based polymeric monoliths for chiral separations and enhanced chemoselectivity for simultaneous chiral separations and enhanced detection in CEC and CEC-MS. In Chapter 6, five amide-linked surfactant-bound monoliths with different chain lengths and head groups (leucine, valine, and phenylalanine) were synthesized and characterized. Enantioseparation of several test compounds was achieved by CEC using the monolithic columns. One of the chiral surfactant, sodium 11-acrylamidoundecanoyl-L-leucinate (SAAUL), was polymerized in aqueous solution under 60Co radiation to form molecular micelle poly-SAAUL. MEKC experiments were carried out with the poly-SAAUL molecular micelle to separate ten cationic chiral compounds. The result was compared with the CEC separation using the AAUL monolithic column. This study is the first comparison of chiral CEC and MEKC with the same surfactant monomer, which has the capability of forming both chiral stationary phase for CEC and chiral pseudophase for MEKC.