Natural products continue to be a major chemical lead matter for drug discovery due to their diverse chemical structures and bioactivities. Clinically significant natural products include anti-cancer and anti-infective compounds and while many more of these compounds show promising bioactivity, their clinical relevance is often limited by toxicity or poor solubility. Combinatorial biosynthesis can be employed to modify existing chemical scaffolds towards reducing these limitations. To fully take advantage of these biochemical tools, it is important to understand the biosynthesis and mechanism of action of the molecules. Saccharides in glycosylated natural products provide specific interactions with cellular targets and are often crucial for a compound’s bioactivity. Genetic engineering of sugar pathways can modify glycosylation patterns leading to the diversification of natural products. Saquayamycins (SQN) H and I are cytotoxic angucycline antibiotics containing five deoxyhexoses including the rare amino sugar rednose. Elucidating the biosynthetic pathway of rednose could add to the arsenal of combinatorial biosynthesis tools for drug development. Our research goal of investigating the rednose biosynthetic pathway was pursued through two specific aims: the identification of the Streptomyces sp. KY 40-1 gene cluster involved in the biosynthesis of SQN H and I (sqn) (specific aim 1), and the validation of the proposed L-rednose biosynthetic pathway up to the glycosyl transfer through enzymatic synthesis of NDP-3,6-dideoxy-L-idosamine (specific aim 2). The sqn gene cluster revealed deoxysugar biosynthetic genes that could be used to alter glycosylation patterns to generate novel compounds while the enzymatic synthesis afforded novel genetic engineering tools to generate novel TDP-deoxysugars that could be used to diversify compounds such as aminoglycosides to circumvent resistance mechanisms. The first step to generate TDP-glucosamine enzymatically was accomplished, however later steps were unsuccessful. The aureolic acid mithramycin (MTM) was recently tested in clinical trials for Ewing sarcoma following the discovery of MTM as a potent inhibitor of the oncogenic transcription factor EWS-FLI1 present only in Ewing sarcoma cells It is understood that MTM binds the minor groove of G/C rich DNA as an Mg2+-coordinated dimer disrupting transcription of proto-oncogenes; however, the DNA recognition rules were not completely understood, making further interrogation of MTM’s DNA binding preferences necessary. This research goal of further understanding the mechanism of action for MTM was approached through two specific aims: the investigation of the dimerization of MTM (specific aim 3), and the investigation of MTM’s DNA binding preferences (specific aim 4). This work established that MTM and its biosynthetic precursor premithramycin B (PreMTM B), and several MTM analogues with modified 3-side chains: mithramycin SDK (MTM SDK), mithramycin SA tryptophan (MTM SA-Trp), and mithramycin SA alanine (MTM SA-Ala) dimerize even in the absence of DNA under physiologically relevant conditions. The study also demonstrated that modification of the 3-side chain modulates DNA binding affinity of MTM analogues, established a minimum MTM binding site on DNA, and revealed MTM DNA recognition is driven by direct (sequence) and not indirect (conformation) readout laying the foundation for subsequent research based on the interaction between MTM, DNA, and the oncogenic transcription factor EWS-FLI1 in the rational design of new MTM analogues for the treatment of Ewing sarcoma.

Metallo-supramolecular hydrogels have interesting dynamic properties for many applications. We report a simple method for synthesizing copper-based polymer hydrogels made from nontoxic poly(methyl vinyl ether-alt-maleic anhydride) (PVM-alt-MA) in the absence or presence of added dicarboxylates, such as adipate and terephthalate. We utilize metal-polycarboxylate backbone and carboxylate spacer ligands between polymers strands engineered via non-covalent metal ion coordination. Rheological measurements revealed that the mechanical stability of the hydrogels was enhanced by the addition of supplementary dicarboxylate ligands. The optimal ratio of polymer to dicarboxylate to Cu2+ was 10:4:2.5. Our scanning electron microscope (SEM) and Cryo-SEM imaging and physical adsorption measurements revealed the formation of pores. The Brunauer–Emmett–Teller (BET) surface area of the dried hydrogels increased from 177.96 m2 g−1 in a dried hydrogel without added dicarboxylate to 646.90 and 536.44 m2 g−1 with the addition of adipate and terephthalate, respectively. The pore volume increased as well. Separation of CO2 from post-combustion flue gases is important for environmental and economic sustainability. The PVM-alt-Na-MA:adipate:Cu2+ hydrogels are promising material for post-combustion CO2 separation. At normal conditions (298 K and 1 bar), the PVM-alt-Na-MA:adipate:Cu2+ hydrogel samples with 10:4:2.5 ratio, showed notable CO2/N2 selectivity of 78.46 and a high CO2/CH4 selectivity reaching 26.09 at 1 bar. Additionally, we investigated in detail the effect of transition metal ion on the rigidity and structure of hydrogels using Al3+, Fe3+, Cu2+, Ni2+, Zn2+, and Co2+. We also studied the effect of using tricarboxylate spacer ligands such as nitrilotriacetic (NTA) and trisodium citrate or tetracarboxylate such as ethylenediaminetetraacetic acid (EDTA). It is important to mention that one of the main advantages of our facile synthesis method is being simple and can be scaled up for commercial applications. For scaling up the synthesis of hydrogels, we utilized a filling machine that is able to increase the amount of hydrogel aliquots with variable volume. Silver-based hydrogels showed significant antibacterial activity, due to the presence of silver nanoparticles. We utilized a filling machine for application of amorphous wound dressing. The optimization of the conditions of the filling enabled us to scale up the synthesis and the filling process.

Modelling of interactions of proteins and peptides with metal ions Ondrej Gutten - Diploma thesis Keywords: Metalloproteins, metal ion selectivity, in silico prediction Abstract: An approach for in silico prediction and estimation of selectivity properties of metal-binding peptides is suggested. An in-depth analysis is performed to disclose the justifiability and limitations of this approach. The study is divided into three parts. First part investigates the soundness of two quantum chemical methods (MP2 and DFT) for their use in the set-up quest. The testing includes comparison with CCSD(T), effect of basis selection, performance of the two methods in geometry optimizations and effect of implicit solvent model. Second part foreshadows the approach of searching for a metal selective peptide by thoroughly investigating the ability of simple representative systems, derived from their metalloprotein templates, to retain the property of interest. Final part describes the initial step of extensive combinatorial approach towards examination of vast number of simple systems that represent metal-binding sites, and which are to be used for prediction of metal-selectivity through exploitation of the described approach and, ultimately, to the de novo design of metalloproteins with desired properties.

This study produced and analyzed shaped tungsten wire tips formed through electrochemical etching. Specifically, the cone length and the radius of curvature of the tip were analyzed. Having the tips move dynamically through an electrolytic solution, such as potassium hydroxide, and tuning the initial starting depth of the tungsten wire along with the dynamic speed of the tungsten wire as it passed throughout the solution allowed various types of tip profiles to be produced. The tip's radius of curvature was able to be reproduced with an accuracy between 88 - 92 %. The method provided would be applicable for the production of various styles of liquid-metal ion source (LMIS) probes and scanning probe microscope (SPM) tips.

This dissertation focuses on developing new methods using the scanning electrochemical microscope (SECM) to produce chemical concentration maps of different chemical species on various surfaces. Reactive oxygen species (ROS) and transition metal ion maps were generated, indicating the presence or absence of relative types of chemical species on the surface. Imaging of both species was based on a modified scanning UME tip and monitoring the change in the tip impedance. 4-Nitrobenzenediazonium tetrafluoroborate was used as the main modifier, and resultant nitrophenyl groups on the modified electrodes were electrochemically converted to aniline to yield the two types of modified electrodes. In the presence of ROS, a permanent change in the impedance accompanies reaction of the surface layer with the ROS, and this change can be used to map the localized reactive species. The spot scanning method was introduced over continuous scanning to enhance the sensitivity. This enhanced method generated a more effective method to map ROS compared to the diAC/dxmajor image in the continuous scanning method. Images obtained by this sacrificial method show that alternating current SECM (AC-SECM) can be used to map ROS on a surface. The capacitive change gives direct indication of the concentration of these highly reactive species. Transition metal ions showed a partially reversible adsorption with aniline-modified electrodes. Localized concentrations of buffered copper and nickel divalent cations were generated by pumping through a micro-capillary embedded in a substrate. Copper and nickel ions on these substrates were mapped successfully. A solution of calcium ions was used as the negative control. Biased nickel, copper, and lead wire-embedded substrates were line scanned to validate these results. An aniline-modified electrode was placed away from the metal wire and the time taken for metal ions to reach the electrode tip was measured after a voltage pulse. These data were compared with calculated diffusion times. Both systems were optimized using the medium pH, scan rates, and tip potentials. AC-SECM coupled with modified electrodes showed the capability of mapping both ROS and some transition metal ions semi-quantitatively.

No description available.

In an electrochemical sensor, the sensing performance is mainly dependent on the mass transport of the analyte towards the working electrode-electrolyte interface and working electrode properties. Carbon nanomaterials like carbon nanotubes are widely employed to modify the working electrode properties for sensitive detection. A simulation model is formulated to investigate the effects of modifying a planar bare electrode with carbon nanotubes on electrochemical detection of fenitrothion (FT, an organophosphate). The model revealed that porous electrodes caused the change in mass transport regime and influenced FT’s electrochemical response. The results aided in understanding the influence of the porous electrode on analyte detection and thus assisted in the fabrication of an ultrasensitive electrochemical sensor. Simulation supported synthesis of a highly sensitive ink to produce highly porous and electrocatalytic electrodes. Activated carbon (AC) possesses high porosity and surface area, but they suffer from lower electrical conductivity. To enhance their conductivity, AC was co-doped with nitrogen and sulfur. Multiwalled carbon nanotubes were incorporated to further improve their porosity and electrocatalytic properties. The synthesized nitrogen-sulfur co-doped activated carbon coated multiwalled carbon nanotube (NS-AC-MWCNT) ink produced highly porous electrocatalytic electrodes. The sensor revealed a 4.9 nM limit of detection (LOD) under optimized conditions. However, it failed to overcome the enzymatic sensors’ performances. The ultrasensitive performance was achieved by incorporating a detecting agent in the ink that instilled analyte capture ability. Metal oxides like ZrO2, MnO2, and MgO possessed affinity towards organophosphate (fenitrothion), heavy-metal ion (lead), and nutrient (nitrite). Metal oxides were modified with 3,4-dihydroxylbenzaldehyde (DHBA) – Chitosan (CHIT) to produce well dispersed and uniformly coated stable electrodes. The ZrO2-DHBA-CHIT/NS-AC-MWCNT sensor achieved a remarkable limit of detection of 1.69 nM for FT. The sensor's performance exceeded the enzymatic-based sensors. The commonly found chemical interferents had negligible interference. The sensor produced reliable and satisfactory performance in lake and tap water. The MnO2-DHBA-CHIT/NS-AC-MWCNT/GCE and MgO-DHBA-CHIT/NS-AC-MWCNT/GCE sensors produced an enormous improvement in the sensor performance compared to unmodified electrodes for lead and nitrite detection. The preliminary results on detecting other pollutants like lead and nitrite showed the importance of the methodology in providing a platform for a new class of metal oxide-based sensors. / Thesis / Doctor of Philosophy (PhD) / The growing population and rapid industrial development are affecting the water quality worldwide. The major water pollutants are organophosphates, heavy metal ions, and nutrients. These water pollutants are harmful, and their bioaccumulation poses a major health concern. In the USA alone, water quality issues are predicted to cost $210 billion annually. Therefore, sensors to detect water pollutants are developed to monitor their environmental footprints. Electrochemical sensors are popularly used to detect water pollutants owing to their low-cost and high sensitivity. The objective of this dissertation was to fabricate highly sensitive and specific electrochemical sensors to detect organophosphate (e.g., fenitrothion, FT), heavy metal ion (e.g., lead), and nutrient (e.g., nitrite). The sensors were fabricated with ink based on nanomaterials like carbon nanotubes and detecting agents like metal oxides. The fabricated sensors achieved very high sensitivity and specificity and can detect water pollutants in lake and tap water.

No description available.

No description available.

Alternating copolymers represent a special class of copolymers in which the two comonomers copolymerize in a regular alternating sequence along the polymer chain. Of particular interest in our group are the stilbene-maleic anhydride/maleimide alternating copolymers. These copolymers possess sterically congested backbones and precisely placed functional groups arising from the strictly alternating copolymerization. The research in this dissertation is focused on the synthesis, characterization, and potential application of functionalized copolymers that contain semi-rigid alternating copolymer sequences. The fluorescence properties of a series of non-conjugated, tert-butyl carboxylate functionalized alternating copolymers were investigated. Extraordinarily high fluorescent intensity with excellent linearity was observed for the di-tert-butyl group-containing stilbene and maleic anhydride alternating copolymer in THF. We attributed the origin of the strong fluorescence to the “through space” π – π interactions between the phenyl rings from the stilbene and C=O groups from the anhydride. The fluorescence was maintained when the copolymer was deprotected and hydrolyzed and the resulting carboxylic acid-functionalized copolymer was dissolved in water at neutral pH. The tert-butyl carboxylate functionalized alternating copolymer sequences were incorporated into highly crosslinked polymer networks using suspension polymerization. After removing the tert-butyl groups by acidic hydrolysis, the surface area of the networks increased significantly. Using this facile two-step strategy, we were able to achieve nanoporous polymers with BET surface area up to 817 m2/g and carboxylic acid-functionalized surfaces. The BET surface area of deprotected polymers increased with increasing crosslinking density, and the stilbene-containing polymers showed systematically higher BET surface area than the styrene-containing polymers due to the stiffness of the alternating sequences. The resulting nanoporous polymers have potential to be employed as solid sorbents for CO2. The same tert-butyl carboxylate functionalized alternating copolymer sequences were also incorporated into microgels via miniemulsion polymerization. The miniemulsion technique ensured the successful synthesis of microgels with ~100 nm diameter using solid stilbene and maleimide monomers. The resulting tert-butyl carboxylate-containing microgels were converted into carboxylic acid-containing aqueous microgels by acid hydrolysis. These aqueous microgels showed good and reversible lead and copper ion adsorption capacities. Amine-functionalized nanoporous polymers were synthesized by the post-modification of highly-crosslinked divinylbenzene-maleic anhydride polymers. High amine-contents were achieved by covalently attaching multiamines to the acid-chloride functionalized polymer surface. The resulting polymers showed medium to high BET surface areas (up to 500 m2/g) and high CO2 capture capacities. / PHD