Browsing by Subject "Chemistry, General"

Cellular damage due to oxidative stress is implicated in a wide variety of conditions including degenerative diseases like Alzheimer's and Parkinson's Diseases. One source of oxidative stress is the interaction of redox-active metals such as copper and iron with hydrogen peroxide to produce hydroxyl radicals. Preventing metal-induced oxidative stress by metal chelation is one potential approach to treat some of these diseases, but there remain significant challenges in designing chelators that target damaging metals while not disturbing healthy metal ion distribution.

To overcome this challenge, prochelators that are responsive to conditions of oxidative stress have been introduced. By designing ligands that only bind metal ions in the presence of oxidants, damaging metals can be bound and removed while not perturbing the metals necessary for cell function. Masking the phenol of a chelator with a boronic ester creates a prochelator that has little to no affinity for metal ions until exposure to H₂O₂ converts the prochelator to the chelator, which is then available to bind metal ions. Described here is the development of boronate-based prochelators that react with H₂O₂ to produce chelating agents of variable denticity, ranging from 2 to 6.

Quinoline boronic acid pinanediol ester, or QBP, is a new bidentate prochelator introduced here that reacts with H₂O₂ with a rate of 0.22 M-1s-1 to produce 8-hydroxyquinoline, a known metal-binding agent. Results in Chapter 2 show that QBP can be activated in vitro under conditions that mimic early Alzheimer's Disease pathology where copper, amyloid beta peptide, and ascorbic acid exacerbate formation of reactive oxygen species. QBP does not bind metal ions, nor does it disaggregate metal-promoted amyloid beta peptide aggregates. However, the released 8-hydroxyquinoline sequesters copper from amyloid beta and both diminishes further formation of reactive oxygen species and inhibits further aggregation of amyloid-beta.

The syntheses and crystal structures of hexadentate prochelators are described in Chapter 3, along with their rates of oxidation in response to hydrogen peroxide exposure and their ability to protect against hydroxyl radicals formed in vitro by iron (or copper), ascorbic acid, and hydrogen peroxide. The hexadentate chelators are based on a tripodal architecture in which three phenol moieties are linked via nitrogens on three alkyl arms to a central nitrogen to provide an N₃O₃ donor set for metal complexation. Of three prochelator/chelator pairs prepared, the pair (trenBsalam/trensalam) with amine linkages was deemed most suitable for potential biological studies. The prochelator trenBsalam oxidizes at a rate of 0.72 M-1s-1 to produce the chelator trensalam in the presence of hydrogen peroxide. The transition metal coordination chemistry and metal ion affinities of trensalam were further studied in Chapter 4 by x-ray crystallography, UV/Vis spectroscopy and cyclic voltammetry.

The response of a series of bidentate prochelators to various oxidants, including hydrogen peroxide, superoxide, peroxynitrite and hypochlorite, was evaluated by UV/Vis spectroscopy in Chapter 5. Varying the diol that is appended to the boronic ester results in hydrogen peroxide oxidation rates ranging from 0.018 to 1.27 M-1s-1. Lastly, the stability of different boronic acid and diol combinations was probed by spectroscopic techniques and indicate that boronic esters formed with pinanediol form the most stable prochelators under physiological conditions.

The goal of this work is to develop novel molecular systems, functionalization techniques, and data collection routines with which to study the binding of immobilized cognate binding partners. Our ultimate goal is the routine evaluation of thermodynamic parameters for immobilized systems through interpretation of the variation of the binary probability of binding as a function of soluble ligand concentration. The development of both data collection routines that minimize non-specific binding and functionalization techniques that produce stable ordered molecular systems on surfaces are of paramount importance towards achievement of this goal. Methodologies developed here will be applied to investigating the thermodynamics of multivalent systems.

In the first part of this work, the effect of contact force on molecular recognition force microscopy experiments was investigated. Increased contact forces (>250 pN) resulted in increased probabilities of binding and decreased blocking efficiencies for the cognate ligand-receptor pair lactose-G3. Increased contact force applied to two control systems with no known affinity, mannose-G3 and lactose-KDPG aldolase resulted in non-specific ruptures that were indistinguishable from those of specific lactose-G3 interactions. Thus, it is essential to design data collections routines that minimize contact forces to ensure that ruptures originate from specific, blockable interactions.

In the second part of this work we report the first example of the preparation of stable self assembled monolayers through hydrosilylation of a protected aminoalkene onto hydrogen-terminated silicon nitride AFM probes and subsequent conjugation with biomolecules for force microscopy studies. Our technique can be used as a general attachment technique for other molecular systems.

In the third part of this work we develop novel molecular systems for tethering oriented vancomycin and its cognate binding partner L-Lys-D-Ala-D-Ala to surfaces and AFM tips. Unbinding experiments demonstrated that traditional methods for forming low surface density amine layers (silanization with APTMS and etherification with ethanolamine) provided molecular constructs which displayed probabilities of binding that were too low and showed overall variability too high to use for probabilistic evaluation of thermodynamics parameters. Instability and heat-induced polymerization of APTMS layers on tips and surfaces also prohibited their utility. Formation of Alkyl SAMs on silicon provides a more reliable, stable molecular system anchored by Si-C bonds that facilitates attachment of vancomycin and is capable of withstanding prolonged exposure to heated organic and aqueous environments. It follows that covalent immobilization of KDADA to silicon nitride AFM tips via Si-C bonds using hydrosilylation chemistry will be similarly advantageous. These methods offer great promise for probabilistic evaluation of thermodynamic parameters characterizing immobilized binding partners and will permit unambiguous determination of the role of multivalency in ligand binding, using an experimental configuration in which intermolecular binding and aggregation are precluded.

Human and yeast analogs of a high affinity copper transport protein (hCtr1 and yCtr1) have been implicated in the transport of platinum-based drugs in cells. Using liquid chromatography and mass spectrometry, we have compared the binding interactions between cisplatin, carboplatin and oxaliplatin with synthetic peptides corresponding to the methionine-rich regions of hCtr1 that encompass amino acid residues 7-14 (MGMSYMDS) and 39-46 (MMMMPMTF). Incubation of cisplatin, cis-diamminedichloroplatinum(II), with a peptide species containing a single methionine results in the loss of 1 or 2 chloride ligands of cisplatin upon coordination of the Pt center to the thioether of methionine and a backbone amide. The interactions of cisplatin and carboplatin with Met-rich motifs containing three or more methionines result in removal of the carrier ligands of both platinum complexes. These results imply that the interaction of cisplatin with methionine-rich regions of hCtr1 alters the drug in such a way that it is no longer in its active form. In contrast, in the presence of a peptide corresponding to residues 39-46 containing 5 methionines, oxaliplatin retains its cyclohexyldiamine ligand upon platinum coordination to the peptide. This result indicates that should oxaliplatin utilize hCtr1 to enter a cell, the drug could remain in its active form.

In an effort to exploit the interactions of Pt(II) species with methionine, a second project focused on the development of a fluorescent Pt(II) species that selectively coordinates methionine-rich motifs. The syntheses of multinuclear Pt(II) and Ru(II)-Pt(II) complexes containing 2,3-bis(2-pyridyl)pyrazine (dpp) as a bridging ligand were carried out and their coordination to sulfur containing amino acids and bovine serum albumin were explored using UV-Vis and Fluorescence Spectroscopy techniques. The coordination of methionine, cysteine and histidine to [Ru(II)[(Pt(II)Cl₂dpp))₃] results in the removal of the Pt(II)Cl2 moieties from [Ru(II)(dpp)₃]. As a consequence, dpp should not be incorporated into a Pt(II) species for the development of a met-specific fluorescent Pt(II) complex.

The discovery of interactions of Pt(II) species with methionine-rich portions of proteins has opened the door for the development of Pt(II) fluorescent species that could potentially coordinate to proteins with specificity for methionine-motifs. The syntheses of multinuclear Pt(II) complexes, using 2,3-bis(2-pyridyl)pyrazine (dpp) as a bridging ligand were carried out and their coordination to sulfur containing amino acids and bovine serum albumin were explored using UV-Vis and Fluorescence Spectroscopy techniques.

Nanostructures are being intensely studied due to unusual material properties and simple scaling concerns in the microelectronics industry. Fabricating useful nano-scale structures and devices, either by arranging existing nanoparticles such as carbon nanotubes or by manipulating bulk materials into nanometer-scale geometries, is a challenging prospect. One promising approach is to generate a nanometer-scale pattern and transfer that geometry into another material. The research described in this dissertation concerns the fabrication of nanometer-scale patterns, by Atomic Force Microscope-based methods and Electron Beam Lithography, on planar surfaces and the transfer of those patterns into functional materials. Differences in surface energy were used to guide the growth of bulk conducting polymer along predefined nano-scale patterns. Carbon nanotubes were assembled into an ordered and continuous material with no guidance and used to lithographically write silicon oxide nanopatterns on a silicon surface. Finally, the two previous projects were combined, and surface energy patterns were used to guide the deposition of dense carbon nanotube bundles along a planar substrate.