Browsing by Subject "Zinc"

Histatin-5 (Hist-5) is a polycationic, histidine-rich antimicrobial peptide with potent antifungal activity against the opportunistic fungal pathogen Candida albicans. Hist-5 can bind metals in vitro, and metals have been shown to alter the fungicidal activity of the peptide. The goal of this work is to gain insight into the role of metals in the biological activity Hist-5. Toward this goal, we developed and characterized a novel fluorescently labeled Hist-5 peptide (Hist-5*) to visualize changes in internalization and localization of the peptide in fungal cells as a function of metal availability in the growth medium. Here, we provide evidence for Zn-modulated antifungal activity of Hist-5 in which the availability of Zn2+ in the surrounding environment inhibits Hist-5 cellular uptake and cidality. Cellular growth assays revealed a concentration-dependent inhibitory effect of Zn2+ on Hist-5 antifungal activity. Imaging by confocal microscopy showed that equimolar concentrations of Zn2+ kept the peptide localized along the cell periphery rather than internalizing, thus preventing cytotoxicity and membrane disruption. We found that modulation of extracellular Zn2+ concentration by metal chelating molecules or proteins reversed Zn-induced surface adhesion of Hist-5, leading us to propose a dynamic role for Zn2+ as an inhibitory switch to regulate Hist-5 fungicidal activity. We next present data to support the hypothesis that Hist-5 interacts with intracellular Cu to increase the fungicidal activity Hist-5. Combined fluorescence spectroscopy and microscopy experiments showed reversible Cu-dependent quenching of Hist-5* fluorescence, indicating a direct interaction between Hist-5 and intracellular Cu. X-ray fluorescence microscopy images revealed peptide-induced changes to cellular Cu distribution and cell-associated Cu content. Finally, we present progress towards expanding the scope in which we understand and assess Hist-5 biological activity by investigating the activity of the peptide under biologically relevant conditions and testing its fungicidal activity against other fungal species.

Aminoarenes are common structural features in pharmaceuticals and biologically relevant scaffolds, motivating continued development of strategies to facilitate their synthesis. Canonical approaches to aryl amination rely upon nucleophilic N–H bond precursors. Nitrogen–heteroatom bonds possess versatile reactivity and can enable synthetically attractive approaches to functionalized aminoarenes. Towards this, we proposed the use of nitrogen–heteroatom bonds in both bond insertion by reactive aryne intermediates and as electrophilic amination reagents in the net C–H amination of arenes via zinc-amide mediated H–Zn exchange.

Herein, we describe the development of nitrogen–heteroatom bond enabled strategies to access functionally diverse aminoarene products. Aryne insertion of N-chloro, -bromo, and even -iodoamines was achieved in moderate yields and excellent regioselectivity to provide direct access to ortho-haloaminoarene motifs. This approach employs ortho-trimethylsilyl aryltriflates and simple fluoride salts as a mild platform for in situ formation of reactive arynes, affording functionally complex aminoarene products in a single transformation. Access to aminoarenes from ubiquitous heteroaryl and aryl C–H bonds was also achieved via copper-catalyzed electrophilic amination with O-benzoylhydroxylamines, mediated by initial H–Zn exchange from highly hindered amide-zinc complexes. In addition to well-established Zn(TMP)2 and Zn(TMP)Cl•LiCl, we developed LiTMP0.1Li[ZnEt2(TMP)] as part of a general ortho-directed C–H zincation/amination strategy. Using O-benzoylhydroxylamines and a copper catalyst to affect electrophilic amination, this zincate base permitted access to a broad scope of heteroarene and arene substrates with varied ortho-directing group functionalities. The improved access to aminoarene products enabled by these methods was then demonstrated through the study of functionally selective dopamine receptor ligands, which yielded valuable information on the complex SAR and SFSR of D2 and D3 receptors and their signaling pathways.

The successful development of strategies to access highly functionalized aminoarene scaffolds provides valuable tools for drug discovery. The methods presented here expand access to diverse aryl amine motifs and will contribute to the development of novel small molecule biochemical tools and therapeutics.

Nanomaterials often exhibit enhanced reactivity relative to their larger colloidal counterparts because of the high specific surface area and number of imperfections on the crystal lattice at the nanoscale. Management of ecosystems, remediation of contaminated waters, and assessment of the potential risks from the industrial use on nanomaterials requires an understanding of the environmental factors that control the reactivity and bioavailability of natural and manufactured nanomaterials. Dissolved organic matter (DOM) acts as a moderator of reactivity and bioavailability for dissolved and particulate moieties in natural waters. DOM consists of a range of low and high molecular weight species that are complex and heterogeneous. It has been historically categorized based on operational definitions, rather than physical properties. In order to understand the effect of DOM on nanomaterials, there is an urgent need for information regarding specific properties of DOM, such as ligand groups.

The goal of this research was to study how cysteine, a low molecular weight metal-binding ligand, affects the composition and reactivity of nanoparticulate zinc sulfide and metallic silver. Zinc sulfide was used as a representative of nanoparticulate metal sulfide which occurs naturally in sulfidic environments. Metallic silver nanoparticles were also studied because of its wide use in consumer products. Both types of nanomaterials contain metal constituents (zinc and silver) that are expected to strongly bind to sulfhydryl-containing ligands (such as cysteine) in the environment. Serine is structurally similar to cysteine, with the only difference of a hydroxyl group in the place of the sulfhydryl group of cysteine. Therefore, serine was used for comparison as a hydroxyl-containing analogue to cysteine.

The aggregation kinetics of zinc and other metal sulfide nanoparticles in the presence of cysteine and serine were investigated using dynamic light scattering. Cysteine decreased aggregation rates of the particles, while serine had no effect on their aggregation behavior. Further experiments revealed that the mechanism of stabilization occurred through the adsorption of cysteine on zinc sulfide, which induced electrostatic charge on the particles surface. A direct link was established between the amount of cysteine sorbed and attachment efficiency, an indicator of the tendency of particles to aggregate. These results shed light on discrepancies in the literature between metal sulfide precipitation experiments conducted in our lab and work on the formation and aggregation of zinc sulfide nanoparticles on biofilms of sulfate reducing bacteria.

The early-stage growth and aggregation kinetics of zinc sulfide nanoclusters in the presence of cysteine was studied in detail using a suite of complementary techniques. Growth and aggregation experiments have been traditionally difficult to conduct due to instrumental precision issues, but newly developed analytical tools and software products have made it possible to study the early-stage formation of nanoclusters. Experiments with small angle X-ray scattering, X-ray diffraction, dynamic light scattering, and X-ray absorption spectroscopy at the extended fine structure range showed that cysteine controlled the growth and aggregation of zinc sulfide nanoclusters. The molar ratio between zinc, sulfide, and cysteine was a determining factor in the precipitation process. When zinc and sulfide were in equimolar concentrations with cysteine, very small nanoclusters of about 2.5 nm formed within 12 hours and aggregated to structures with hydrodynamic diameter larger than 100 nm. When cysteine was in excess of zinc and sulfide, aggregation was held to a minimum, but monomer nanoclusters were able to grow to about 5 nm in 12 hours. Overall, these results indicate the importance of thiol ligands on the monomer size, extent of aggregation, and aggregate structure of zinc sulfides.

The effect of metal ligands on metal bearing particle surfaces is of particular interest for manufactured nanoparticles, because they are typically coated with an organic coating during the production process. These coatings are sorbed on the particles surface and are likely to interfere between the metallic surface and the ligand. Dissolution experiments using citrate and polyvinylpyrrolidone (PVP) coated zero valent silver nanoparticles in the presence of cysteine and serine showed that cysteine dissolved both types of particles, while serine did not. Dissolution rates depended on the aggregation state of the particles exposed to cysteine. As indicated by zeta potential and adsorption measurements, cysteine replaced the coating on the particles surface and altered their aggregation pattern. X-ray absorption spectroscopy near the absorption edge showed partial oxidation of silver and formation of Ag(+I)-sulfur bonds, indicating that the thiol group in cysteine formed chemical bonds with oxidized surface silver atoms. A comparison between the two coatings showed that citrate coated particles dissolved approximately three times faster than PVP coated particles. Overall, these results show that metal binding ligands can drastically change the fate of manufactured silver nanoparticles in the environment and that this effect is moderated by surface coatings.

The results of this study suggest that cysteine, a metal binding ligand was able to induce and control transformations, such as growth, aggregation, dissolution, and surface reactivity of zinc sulfide and metallic silver nanoparticles. Cysteine adsorbed on metal sites on both ZnS and Ag particles, inducing changes on their surface charge. Aggregation of ZnS particles was slowed because of a net decrease in zeta potential compared to the bare particles. On the contrary, cysteine enhanced the aggregation of Ag particles, by replacing the citrate and PVP coatings on the particles surface. Finally, the cysteine-Ag(+I) bonds caused strong polarization on the particles surface and lead to the oxidative dissolution of the particles.

Overall, this research provides a better understanding of the fate of natural and manufactured nanoparticles in anaerobic waters, where thiols are present in significant amounts. It may also be used for risk assessment of manufactured nanomaterials and the production of safer and environmentally responsible materials.