Enzyme-Activated Nitric Oxide-Releasing Composite Material for Antibacterial Activity Against Escherichia coli.

Bacterial infections occurring on medical devices are incredibly difficult to treat, highlighting the urgency for progress in developing antibiotics and antibacterial materials. This work describes the preparation of an antibacterial prodrug polymer composite material for use as an antibacterial coating for medical devices to prevent infections. Polyvinyl chloride and polyurethane films are prepared containing a bacterial nitroreductase enzyme-activated diazeniumdiolate that releases nitric oxide (NO), a known potent antimicrobial agent. Characterization of the surface of the composite materials by scanning electron microscopy (SEM) and energy-dispersive X-ray analysis (EDS) reveals that the surface of the materials is composed of high amounts of nitrogen due to incorporation of the NO donor compound, up to 13.2% nitrogen on the surface of the 2.5% w/v diazeniumdiolate composite. NO release from the composite films is observed only after metabolism by a bacterial nitroreductase enzyme isolated from E. coli, demonstrating the prodrug nature of the polymer composite materials. Antibacterial efficacy experiments resulted in up to a 66% reduction in E. coli after exposure to the diazeniumdiolate-composite materials. This work details the first illustration of an antibacterial enzyme-activated NO-releasing polymer, a material with potential application as a medical device coating to prevent device-associated infections and improve patient outcomes.

Synthesis of novel nitroreductase enzyme-activated nitric oxide prodrugs to site-specifically kill bacteria.

The developing antibiotic resistance crisis creates a serious need for new antimicrobial agents. In this work, novel nitroaromatic-protected piperazine diazeniumdiolate (nitric oxide donor) prodrugs are synthesized to release nitric oxide upon enzyme activation to kill bacteria. Antibacterial prodrugs could help reduce side effects due to antibiotics, only releasing the therapeutic where infections are concentrated. The nitroreductase enzyme, which is found almost exclusively in bacteria, reduces the nitroaromatic-protecting group of the synthesized compounds and catalyzes the release of nitric oxide. This paper shows that nitric oxide release from the synthesized compounds only occurs in the presence of a bacteria-derived nitroreductase enzyme, demonstrating the possibility of site-specific delivery of an antibacterial therapeutic. The amount of nitric oxide release is measured at concentrations of 0.01, 0.1, and 1 mM, and is well within antibacterial levels at concentrations of 0.1 and 1 mM. The antibacterial activity of the compounds is demonstrated after exposure of the compounds to Escherichia coli, a nitroreductase-producing bacterial species, leading to up to a 94% reduction in the number of viable bacteria after 24 h at 1 mM concentrations of the prodrug. This study is the first example of an antibacterial diazeniumdiolate prodrug activated by a nitroreductase enzyme, and further demonstrates the possibilities of antibacterial prodrugs.

Sodium-Catalyzed Friedel-Crafts Reactions and Mechanistic Insight.

Sodium tetrakis[3,5-bis(trifluoromethyl)phenyl]borate (NaBArF) demonstrates catalytic activity for Friedel-Crafts addition reactions of phenolic and other aromatic nucleophiles to trans-ß-nitrostyrene. Solubility studies demonstrate a chelating effect between the Na+ cation and the nitro group of trans-ß-nitrostyrene as the basis for catalytic activation. Mechanistic studies are presented, including a comparison with other sodium salts, additive effects, and reaction progress kinetics analysis using 19F NMR spectroscopy.

Fluorescent nitric oxide donor for the detection and killing of Pseudomonas aeruginosa.

The epidemic of multidrug-resistant bacteria calls for the improvement of both detection methods for bacterial infections and methods of treatment. Nitric oxide is a known potent antibacterial agent, but due to its gaseous and highly reactive nature, it is difficult to incorporate into a stable antibacterial compound. In this paper, we synthesize a nitric oxide donor attached to a fluorescent compound, creating a material that can both detect and kill the deadly multi-drug resistant bacteria strain Pseudomonas aeruginosa. Detection occurs through a bacterial enzyme-activated color change, showing a clear and obvious change from blue to yellow under UV light. The synthesized compound spontaneously releases 853 µmol of nitric oxide/g from a 10 mM initial concentration. Antibacterial efficacy studies after exposing Pseudomonas aeruginosa to a 10 mM dose of the synthesized compound show a 55-75% reduction in bacteria after 24 hours. This work is the first instance of a small molecule dual-function material that can both detect and kill bacteria.

Derivatization of Dextran for Multiply Charged Ion Formation and Electrospray Ionization Time-of-Flight Mass Spectrometric Analysis.

We present the use of a simple, one-pot derivatization to allow the polysaccharide dextran to carry multiple positive charges, shifting its molecular weight distribution to a lower m/z range. We performed this derivatization because molecular weight measurements of polysaccharides by mass spectrometry are challenging because of their lack of readily ionizable groups. The absence of ionizable groups limits proton abstraction and suppresses proton adduction during the ionization process, producing mass spectra with predominantly singly charged metal adduct ions, thereby limiting the detection of large polysaccharides. To address this challenge, we derivatized dextran T1 (approximately 1 kDa) by attaching ethylenediamine, giving dextran readily ionizable, terminal amine functional groups. The attached ethylenediamine groups facilitated proton adduction during the ionization process in positive ion mode. Using the low molecular weight dextran T1, we tracked the number of ethylenediamine attachments by measuring the mass shift from underivatized to derivatized dextran T1. Using electrospray ionization time-of-flight mass spectrometry, we observed derivatized dextran chains ranging from two to nine glucose residues with between one and four attachments/charges. Our success in shifting derivatized dextran T1 toward the low m/z range suggests potential for this derivatization as a viable route for analysis of high molecular weight polysaccharides using electrospray ionization time-of-flight mass spectrometry. Graphical Abstract ᅟ.