Dissertations / Theses on the topic 'Institute of Organic Chemistry and Biochemistry'

Metal-organic frameworks have a wide range of applications including gas separation, gas capture, catalysis and drug delivery. Due to the in-stent thrombosis of the current drug-eluting stents we propose replacing the toxic polymer with a more biodegradable MOF thin film consisting of MIL-88b. The MIL-88b thin film was formed on functionalized gold through a direct crystallization method and was confirmed using x-ray diffraction (XRD) and Fourier- transform infrared spectroscopy (FTIR). Possible ibuprofen encapsulation and elution was confirmed through FTIR and UV-VIS spectroscopy. The MIL-88b thin film was also formed on medical grade stainless steel to mimic conditions of the current DES. The surface area, using N₂ gas isotherm at 770K, of MIL-88b and MIL-53 was compared to validate the favorable porosity for drug delivery application.

The synthesis of complex carbohydrates containing sialic acid is very challenging by chemical methods, in part due to the competing elimination reaction, as well as poor stereocontrol. One major disadvantage in the field is the limited knowledge of the mechanism of sialylations. The mechanism is believed to go through an oxacarbenium ion intermediate. Recent findings suggest the existence of an all-axial conformation of the oxacarbenium ion that might be important in controlling the overall efficiency of sialylations. As a part of a general research goal to gain a better understanding of sialylations, we describe herein the synthesis of a C-3 deuterium-labeled substrate that might give insight on the existence of such conformation.

Recently, it has been shown that a decrease in activity of acetylcholinesterase (AChE) in AD patients is compensated by an increase in butyrylcholinesterase (BuChE) activity. Therefore, BuChE also becomes a significant target in the treatment of cognitive loss associated with Alzheimer’s disease. While the majority of drug development so far has centered on AChE inhibitors in order to increase the acetylcholine level in AD patients, the development of specific and potent BuChE inhibitors have also begun to attract attention. We recently synthesized and assayed a library of bisthiophosphates as potential BuChE inhibitors. To evaluate if the analogs were selective for BuChE, their inhibitory properties against both BuChE and AChE were determined.

Site-directed mutagenesis provides a powerful tool in the study of enzyme function. Residues suggested to be important for catalysis can be readily mutated and the energetic effects measured. However, the limited repertoire of naturally occurring amino acids constrains the substitutions that can be made. To obtain a deeper understanding of how enzymes work requires using unnatural amino acids to systematically perturb enzymatic residues. For example, hydrogen bonds in an ‘oxyanion hole’ are a common feature of enzyme active sites and often suggested to be important for catalysis. However, water can form hydrogen bonds, so for enzyme-mediated hydrogen bonds to be catalytic the energetics of these hydrogen bonds must be different than those made with water. A previous study in the enzyme ketosteroid isomerase (KSI) used a series of fluorotyrosine analogs to perturb the pK_a of the tyrosine hydrogen bond donor and results suggested a modest catalytic contribution of oxyanion hole hydrogen bonds. However, challenges in synthesis limited the set of fluorotyrosine analogs used. To overcome these challenges and extend the series of fluorotyrosines used in enzymatic studies, we developed an approach to selectively incorporate fluorotyrosines in peptides using silyl-based protecting groups. The fluorotyrosine must be protected on the amino and phenol groups and then incorporated in a peptide using solid phase synthesis. More so, the protection chemistry must be friendly in such a way that it does not have drastic side effects on any other part of the system. We tested the stability of silyl groups including TBDMS-Cl, TIPS-Cl, and TBDPS-Cl for their use in peptide synthesis. With the doubly protected fluorotyrosines, solid phase peptide synthesis will occur in order to place the new tyrosine into the protein of interest where these analogs will be used to investigate the energetics of enzymatic hydrogen bonds.

This dissertation describes metabolic engineering of cyanobacterium Synechococcus elongatus PCC7942 as a photosynthetic host for the conversion of CO₂ into 2,3-butanediol. Current advances in pathway design, genetic tool development, and yield improvement are described (Chapter 1). A pathway for the synthesis of 2,3-butanediol is designed based on collective concepts of pathway strength, robustness, and irreversibility, and extensively tested through the generation of mutants (Chapter 2). This pathway is then optimized through modulation of translation by combinatorial mixing of ribosome binding sites (Chapter 3). Finally, photosynthetic productivity is investigated through expression of an exogenous pathway targeting every step between fixation and product (Chapter 4). All materials and methods are given separately for easy reference (Chapter 5).

The relevance of protein-based biopharmaceuticals has increased dramatically in the past decades and a variety of products are now available for human therapy. Antibodies in particular are currently the most heavily consumed protein therapeutics, with a current market volume expected to reach 1 trillion US$ in 2015 and a compound annual growth rate (CAGR) of 3-6%. Meeting the increasing demand for these therapeutics at lower prices while complying with increasingly stringent regulatory environments, calls for the development of new technologies and platform approaches for efficient downstream protein purification. Extended use of affinity chromatography holds great promise in meeting the urgent demand for affordable high-quality biological products. This technology, however, is still dependent on the use of biological ligands, such as Protein A, Protein G, and Protein L, that have significant issues associated with their high cost, harsh elution conditions, narrow specificity, low chemical stability, and immunogenicity in patients if they leach into the product stream. Small, robust, synthetic ligands may offer an effective alternative to protein ligands. Peptides in particular combine levels of affinity and specificity similar to those of biological ligands with high chemical and biochemical stability, broader specificity, low immunogenicity and ease of synthesis that can reduce costs.

The work in this thesis aims to discover and characterize novel peptide ligands to produce efficient, robust, and affordable affinity adsorbents for improved downstream purification of biologics. Two main areas have been investigated: (a) the development of linear hexapeptide – based adsorbents for the purification of human antibodies and (b) the design and screening of novel libraries of cyclic peptides for the discovery of novel ligands.

The research conducted on the characterization and development of competitive peptide-based affinity adsorbents comprises: (a.1) testing existing peptide ligands for the purification of antibodies from a variety of sources; (a.2) optimizing the protocol of ligand coupling on chromatographic resins to increase adsorbent binding capacity; (a.3) a method of modification of the resin’s surface chemistry to increase the adsorbent's chemical stability in harsh alkaline conditions; and (a.4.) a combined computational and chemical strategy for the design of protease-stable peptide ligands. The resulting peptide affinity adsorbents compete well with advanced Protein A – based adsorbents in terms of product yield and purity, dynamic binding capacity (~ 50 – 60 g/L), resistance to alkaline cleaning and sanitization, and biochemical stability in the presence of proteolytic enzymes.

In the second part of this work, two methods are presented for the design, synthesis, and screening of libraries of cyclic peptides for the identification of novel affinity ligands. The first method involves the generation and screening of (b.1) a biological mRNA-display library of cyclic peptides, and the second method (b.2) uses a synthetic solid-phase library of “reversible cyclic peptides”. Both libraries have been screened for the identification of ligands for human antibodies. The results of these studies indicate that these libraries are very promising tools for the discovery of robust, selective and affordable peptide ligands.

The methods presented herein offer a new set of tools, not only for affinity ligand discovery, but also for finding new drugs and diagnostic methods. Besides their technological value, these studies also offer insights into the mechanisms of non-covalent interaction that underlie the phenomena of biorecognition and protein activity.

In this work the redox properties of cytochrome c nitrite reductase (CcNiR), a decaheme homodimer that was isolated from S. oneidensis, were determined in the presence and absence of the strong-field ligands cyanide and nitrite. Four hemes per CcNiR protomer are hexa-coordinate with tightly bound axial histidines, while the fifth (active site) has one tightly bound lysine and a distal site that can be open, or contain exogenous ligands such as the substrate nitrite. Controlled potential electrolysis in combination with UV/visible absorption (UV-vis) and electron paramagnetic resonance (EPR) spectroscopies allowed for assignment of all heme midpoint potentials under each set of conditions. The studies show that the active-site heme is the first to be reduced under all conditions. The midpoint redox potential of that heme shifts approximately 70mV to the positive upon binding a strong field ligand such as nitrite or cyanide. When controlled potential electrolysis was carried out in the presence of nitrite, a concerted two electron reduction was observed by UV-vis, and a {Fe(NO)}⁷ reduced product was revealed in EPR. In addition, an asymmetry in ligand binding between active sites was revealed. This information is relevant for the interpretation of planned and ongoing mechanistic studies of CcNiR.

Over-expression, partial purification and characterization of another S. oneidensis multiheme enzyme, known as octaheme tetrathionate reductase (OTR), is also described herein. Though of unknown cellular function, OTR was previously reported to have tetrathionate reductase activity, in addition to nitrite and hydroxylamine reductase activities. The new results indicate that the expression of OTR has no effect on tetrathionate or nitrite reductase activities in the whole cell lysate, and only hydroxylamine reductase activity was substantially elevated in the overexpressing bacteria. OTR was stable in buffered solutions, but substantial activity loss during all attempts at column chromatography was a major obstacle to the complete purification. OTR also proved quite hydrophobic, so possible membrane association should be considered in future attempts to purify this protein.

Finally, this dissertation also reports attempts to improve S. oneidensis' ability to express foreign proteins. Though ideally suited to expressing c-hemes, it proved difficult to express carboxy his-tagged proteins in S. oneidensis because of persistent tag degradation. Attempts to knock out lon protease, a cytoplasmic carboxypeptidase, as well as the result of redirecting ccNiR from the SecA to the possibly more protected signal particle recognition (SRP) secretion pathway, are described.

Iron heme cofactors are single-electron transport moieties that play a crucial role in respiration. While oxygen is the electron acceptor of choice in aerobic atmospheres, microorganisms that live in anaerobic environments utilize other molecules with similarly high reduction potentials. S. oneidensis can utilize numerous terminal electron acceptors, including nitrite, dimethylsulfoxide and even uranium, thanks to a particularly rich array of multi c-heme respiratory proteins. Understanding of how the midpoint potentials and heme arrangements within the proteins influence these exotic respiratory processes is of interest in the fields of bioremediation and fuel development.