Near-infrared (NIR) fluorescence imaging has emerged as an attractive non-invasive approach for direct visualization of diseases which depends on the development of stable, highly specific and sensitive optical probes. The NIR region of the electronic spectrum offers a reduction in the background autofluorescence and an increase in the tissue penetration depth. Cyanine dyes have often been considered promising contrast optic agents owing to their photophysical properties. Herein the synthesis of various penta- and heptamethine benzothiazole cyanine dyes has been described and their in vivo imaging efficacy was determined. Varying functionalities on the benzothiazole aromatic ring and changing substituents on the benzothiazolium nitrogen atom reflected subsequent changes in the imaging pattern and have resulted in the development of promising brain targeting agents.

In the everlasting battle against cancer the development of drugs targeting new therapeutic pathways is of crucial importance. In the attempt to develop new anticancer agents we have synthesized a library of anthraquinone compounds that show selectivity against leukemia. Mechanistic evaluation of the lead compound reveal that this class of compounds achieve their effects through inhibition of MDM2-MDM4 heterodimer and upregulation of the tumor suppressor p53. Computer aided rational design resulted in the development of a number of compounds with activities in the nanomolar range against various cancer cells. Analysis of the physicochemical properties of selected compounds allowed for their evaluation as potential drug candidates. The successful development of non-toxic formulations permits for the further in vivo investigation of the compounds. Click reactions have found wide spread applications in sensing, materials chemistry, bioconjugation, and biolabeling. A number of very useful click reactions have been discovered, which allow for various applications. In bioconjugation applications, the ability to conduct a secondary conjugation will be very useful in, e.g., protein pull down and binding site identification. Along this line, we describe a neighboring group-assisted facile condensation between an aldehyde and a vicinal aminothiol moiety, leading to the formation of benzothiazoles. The conversion is completed within 5 minutes at low micromolar concentrations at ambient temperature. The facile reaction was attributed to the presence of a neighboring boronic acid, which functions as an intramolecular Lewis Acid in catalyzing the reaction. The boronic acid group is compatible with most functional groups in biomolecules and yet can also be used for further functionalization via a large number of well-known coupling reactions.

The Gram-positive pathogens Corynebacterium diphtheriae and Streptococcus pyogenes both require iron for survival. These bacteria have developed sophisticated heme uptake and transport protein machinery responsible for the import of iron into the cell, in the form of heme from the human host. The heme utilization pathway (hmu) of C. diphtheriae utilizes multiple proteins to bind and transport heme into the cell. One of these proteins, HmuT, delivers heme to the ABC transporter HmuUV. The axial ligation of the heme in HmuT was probed by examination of wild-type HmuT and a series of conserved heme pocket residue mutants, H136A, Y235A, R237A, Y272A, M292A, Y349A, and Y349F. Characterization by UV-visible absorption, resonance Raman, and magnetic circular dichroism spectroscopies indicated that H136 and Y235 are the axial ligands in HmuT. Electrospray ionization mass spectrometry was also utilized to assess the roles of conserved residues in contribution to heme binding. The S. pyogenes streptococcal iron acquisition (sia)/heme transport system (hts) utilizes multiple proteins to bring host heme to the intracellular space. Both the substrate binding protein SiaA and the hemoprotein surface receptor Shr were investigated. The kinetic effects on SiaA heme release were probed through chemical unfolding of axial ligand mutants M79A and H229A, as well mutants thought to contribute to heme binding, K61A and C58A, and a control mutant, C47A. The unfolding pathways showed two processes for protein denaturation. This is consistent with heme loss from protein forms differing by the orientation of the heme in the binding pocket. The ease of protein unfolding is related to the strength of interaction of the residues with the heme. Shr contains two NEAT (near-iron transporter) domains (Shr-N1 and Shr-N2) which can both bind heme. Biophysical studies of both Shr-N1 and Shr-N2 indicated a new class of NEAT domains which utilize methionine as an axial ligand, rather than a tyrosine. Thermal and chemical unfolding showed ferrous Shr-N1 and Shr-N2 to be most resistant to denaturation. Shr-N2 was prone to autoreduction. Together, sequence alignment, homology modeling, and spectral signatures are all consistent with two methionines as the heme ligands of this novel type of NEAT heme-binding domain.

Cyclophilins are ubiquitous enzymes that are involved in protein folding, signal transduction, viral proliferation, oncogenesis, and regulation of the immune system. Cyclophilin A is the prototype of the cyclophilin family. We use molecular dynamics to describe the catalytic mechanism of cyclophilin A in full atomistic detail by sampling critical points along the reaction coordinate, and use accelerated molecular dynamics to sample cis-trans interconversions. At these critical points, we analyze the conformational space sampled by the active site, flexibility of the enzyme backbone, and modulation of binding interactions.We use Kramer’s rate theory to determine how diffusion and free energy contribute to lowering the activation energy of prolyl isomerization. We also find preferential binding modes of several cyclophiln A inhibitors, and compare the conformational space sampled by inhibited cyclophilin A to the conformational space sampled during wild-type interactions. We also analyze the mechanism of the next family member cyclophilin B in order to probe differences in enzyme dynamics and intermolecular interactions that could possibly be exploited in isoform-specific drug design. Our results indicate that cyclophilin proceeds by a conformational selection binding mechanism that manipulates substrate sterics, electrostatic interactions, and multiple reaction timescales in order to speed up reaction rate. Conformational space sampled by cyclophilin when inhibited and when undergoing wild-type interactions share significant similarity. Cyclophilins A and B do have notable differences in enzyme dynamics, due to variation in intramolecular interactions that arise from variation in primary structures. This work demonstrates how computational methods can be used to clarify catalytic mechanisms.

Enzymes are ubiquitous in biological systems. They catalyze chemical reactions and are involved in many biochemical processes. The enzyme of interest is Pseudomonas aeruginosa D-arginine dehydrogenase (PaDADH). This flavin-dependent enzyme is composed of approximately 375 amino acid residues and has a broad substrate specificity with D-amino acids. A water recognition motif, observed in roughly 1200 non-redundant protein data bank (PDB) structures, was revealed to be embedded near the active site of PaDADH. This motif coincides with the conformational changes of the enzyme’s gated mechanism. Molecular dynamics simulations were carried out to study the gated properties and structural characteristics of PaDADH in solution. Single amino acid mutations were undertaken to further understand the dynamics of the gated mechanism of this enzyme. In addition, pKa,shift analyses were evaluated to probe for the basic catalytic amino acid residue that is suggested to trigger the catalytic mechanism of PaDADH.

Bacteriophages are viruses that infect bacteria. Phage DNA packaging is one process in the assembly of mature phages that is not well characterized. Elucidating the mechanism of this process would enhance our understanding of the biological significance of the machinery. A previous study suggested that the protein gp74 from bacteriophage HK97 functions as an HNH endonuclease and is required for phage DNA packaging. In this thesis the functionality was assessed for a protein from a phage that replicates under extreme conditions. Our data suggest that the endonuclease activity may not be essential for the role of gp74 in phage DNA packaging.

Dihydroxyacetone phosphate (DHAP)-dependent aldolases have been intensively studied and widely used in the synthesis of carbohydrates and complex polyhydroxylated molecules. However, the strict specificity toward donor substrate DHAP greatly hampers their synthetic utility. We transformed DHAP dependent aldolases mediated in vitro reactions into bioengineered Escherichia coli (E. coli). Such flask-to-cell transformation addressed several key issues plaguing in vitro enzymatic synthesis: 1) it solves the problem of DHAP availability by in vivo hijacking DHAP from glycolysis pathway of bacterial system, 2) it circumvents purification of recombinant aldolases and phosphatase, and 3) it dephosphorylates resultant aldol adducts in vivo, thus eliminating the additional step for phosphate removal and achieving in vivo phosphate recycling. The engineered E. coli strains tolerate a wide variety of aldehydes as acceptor, and provide a set of biologically relevant polyhydroxylated molecules in gram scale. Sialic acids exist in abundance in glycan chains of glycoproteins and glycolipids on the surface of all eukaryotic cells and some prokaryotic cells. Their presence affects the molecular properties and structure of glycoconjugates, modifies their functions and interactions with other molecules. The sialylation status, referring to the expression levels and linkages of sialic acids on the cell surface, is determined by the dynamic balance between sialylation and desialylation (removal of sialic acids). Sialylation is mainly regulated through expression and activity of sialyltransferases. And the mainstream idea attributes desialylation to the sialidases. However, more and more emerging evidences support the existence of ROS/RNS mediated chemical desialylation process under some pathological conditions. We used electrochemical oxidation of sialic acid conjugates to mimic ROS mediated chemical desialylation. Such electrochemical desialylation mimicry reveals that 1) β-linked sialic acid is much more difficult to de desialylated than α-linked sialic acid, 2) electron withdrawing residue and bulky underlying residue can facilitate the desialylation, 3) α- 2,3-linked sialic acid is easier to be desialylated than α-2,6- and α-2,8-linked sialic acid. This information is highly valuable for identifying the ROS species participated in ROS mediated desialylation and unveiling corresponding mechanisms. The mechanism of ROS mediated desialylation was proposed to go through radical decarboxylation.

Lipopolysaccharides (LPS), major virulence determinants in Gram–negative bacteria, are responsible for many pathophysiological responses and can elicit strong immune responses. In order to better understand the role of LPS in host–pathogen interactions and elucidate the immunogenic properties of LPS outer core oligosaccharide, an all α–linked Escherichia coli R3 outer core pentasaccharide was first synthesized with a propyl amino linker at the reducing end. This oligosaccharide was also covalently conjugated to a carrier protein (CRM197) via the reducing end propyl amino linker. An immunological analysis demonstrated that this glycoconjugate can elicit specific anti-pentasaccharide antibodies with in vitro bactericidal activity. These findings will contribute to further exploring this pentasaccharide antigen as a vaccine candidate. Human milk oligosaccharides (HMOs) are a family of diverse unconjugated glycans that exist in human milk as one of the major components. Characterization, quantification and biofunctional studies of HMOs remain a big challenge due to their diversity and complexity. The accessibility of homogenous HMOs library is essential to solve these issues which have beset academia for several decades. In this study, an efficient chemoenzymatic strategy, namely Core Synthesis/Enzymatic Extension (CSEE), for rapid production of diverse HMOs was reported. Based on 3 versatile building blocks and 4 robust glycosyltransferases, a library of 31 HMOs were chemoenzymatically synthesized and characterized by MS and NMR. CSEE indeed provides a practical approach to harvest structurally defined HMOs for various applications. Glycoproteins are extremely important for all life on the planet. Glycoproteins play important roles in various biological processes. Increasing evidences demonstrate that glycosylation of proteins could improve stability of proteins by stabilizing their tertiary structure and protecting them from proteolysis. Besides, glycosylation of proteins could provide targeting effects through glycan-lectin interaction. Furthermore, carbohydrates play crucial roles in humoral immunity in that many sugar epitopes are identified as antigens for antibodies. Glycoprotein could boost strong T cells mediated intercellular immune responses because homogeneous antigens present on the surface of proteins by multivalent bonds. In this study, the three advantages of glycoproteins, namely stabilizing proteins, targeting effects and eliciting immunological response, were extensively explored by broad collaboration with other groups.

In 2007, a gene homolog of CDO encoded by the gene Gm237 in the DUF164 family was identified as cysteamine dioxygenase (ADO). ADO is one of the only known thiol dioxygenases found in mammals. Both ADO and its partner cysteine dioxygenase (CDO) are non-heme iron dependent enzymes that play a crucial role in the biosynthesis of taruine/hypotaurine by insertion of a dioxygen molecule. However, ADO has been overshadowed by CDO as heavy research focus on CDO over the past decade has led to the elucidation of its structure and possible mechanistic properties. In an effort to further understand ADO’s mechanism and regulating role in vivo, this work will be focused on the mammalian hADO and trying to gain further insight on hADO’s structural features via crystallography work. Investigation of the crystallization parameters for hADO has elucidated several potential conditions. Detailed work on these crystallization parameters will be presented.

Micellar electrokinetic chromatography (MEKC), one of the major capillary electrophoresis (CE) modes, has been interfaced to mass spectrometry (MS) to provide high sensitivity and selectivity for analysis of chiral compounds. The research in this dissertation presents the development of novel polymeric glucopyranoside based molecular micelles (MoMs) (aka. polymeric surfactants) and their application in chiral MEKC-MS. Chapter 1 is a review of chiral CE-MS - in the period 2010-2015. In this chapter, the fundamental of chiral CE and CE-MS is illustrated and the recent developments of chiral selectors and their applications in chiral EKC-MS, CEC-MS and MEKC-MS are discussed in details. Chapter 2 introduces the development of a novel polymeric α-D-glucopyranoside based surfactants, n-alkyl-α-D-glucopyranoside 4,6-hydrogen phosphate, sodium salt. In this chapter, polymeric α-D-glucopyranoside-based surfactants with different chain length and head groups have been successfully synthesized, characterized and applied as compatible chiral selector in MEKC-ESI-MS/MS. or the enantioseparation of ephedrines and β-blockers. Chapter 3 continues to describe the employment of polymeric glucopyranoside based surfactants as chiral selector in MEKC-MS/MS. The polymeric β-D-glucopyranoside based surfactants, containing charged head groups such as n-alkyl β-D-glucopyranoside 4,6-hydrogen phosphate, sodium salt and n-alkyl β-D-glucopyranoside 6-hydrogen sulfate, monosodium salt were able to enantioseparate 21 cationic drugs and 8 binaphthyl atropisomers (BAIs) in MEKC-MS/MS, which promises to open up the possibility of turning an analytical technique into high throughput screening of chiral compounds. Physicochemical properties and enantioseparation capability of polymeric β-D-glucopyranoside based surfactants with different head groups and chain lengths were compared. Moreover, the comparison of polymeric α- and β-D-glucopyranoside 4,6-hydrogen phosphate, sodium salt were further explored with regard to enantioseparations of ephedrine alkaloids and b-blockers. The concept of multiplex chiral MEKC-MS for high throughput quantitation is demonstrated for the first time in scientific literature.