Gregory J. Gabriel

Facile Synthesis of Silver Nanoparticles Stabilized by Cationic Polynorbornenes and Their Catalytic Activity in 4-Nitrophenol Reduction

We report the facile one-pot single-phase syntheses of silver nanoparticles stabilized by norbornene type cationic polymers. Silver nanoparticles (AgNPs) stabilized by polyguanidino oxanorbornenes (PG) at 5 and 25 kDa and polyamino oxanorbornenes (PA) at 3 and 15 kDa have been synthesized by the reduction of silver ions with NaBH4 in aqueous solutions at ambient temperature. The four different silver nanoparticles have been characterized by UV-vis spectroscopy, Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), and transmission electron microscopy (TEM) for their particle size distributions. Interestingly, PG stabilizes the silver nanoparticles better than PA as evident from our spectroscopic data. Furthermore, the AgNP-PG-5K (5K = 5 kDa) was found to serve as an effective catalyst for the reduction of 4-nitrophenol to 4-aminophenol in the presence of NaBH4. The reduction has a pseudo-first-order rate constant of 5.50 × 10(-3) s(-1) and an activity parameter of 1375 s(-1) g(-1), which is significantly higher than other systems reported in the literature.

Synthetic Mimic of Antimicrobial Peptide with Nonmembrane-Disrupting Antibacterial Properties

Polyguanidinium oxanorbornene (PGON) was synthesized from norbornene monomers via ring-opening metathesis polymerization. This polymer was observed to be strongly antibacterial against Gram-negative and Gram-positive bacteria as well as nonhemolytic against human red blood cells. Time-kill studies indicated that this polymer is lethal and not just bacteriostatic. In sharp contrast to previously reported SMAMPs (synthetic mimics of antimicrobial peptides), PGON did not disrupt membranes in vesicle-dye leakage assays and microscopy experiments. The unique biological properties of PGON, in same ways similar to cell-penetrating peptides, strongly encourage the examination of other novel guanidino containing macromolecules as powerful and selective antimicrobial agents.

Metal–Ligand-Containing Polymers: Terpyridine as the Supramolecular Unit

New and interesting properties can be obtained from macromolecular architectures functionalized with supramolecular moieties, particularly metal-ligand complexes. Self-assembly, based on the selective control of noncovalent interactions, guides the creation of hierarchically ordered materials providing access to novel structures and new properties. This field has expanded significantly in the last two decades, and one of the most ubiquitous functionalities is terpyridine. Despite its wide-spread use, much basic knowledge regarding the binding of terpyridine with metal ions remains unknown. Here, the binding constants of PEG-substituted terpyridine in relation to other literature reports are studied and a few examples of supramolecular materials from our laboratory are summarized.

Stimuli-responsive polyguanidino-oxanorbornene membrane transporters as multicomponent sensors in complex matrices

We introduce guanidinium-containing synthetic polymers based on polyguanidino-oxanorbornenes (PGONs) as anion transporters in lipid bilayers that can be activated and inactivated by chemical stimulation. According to fluorogenic anion export experiments with vesicles, PGON transporters are most active in neutral bilayers near their phase transition, with EC50’s in the nanomolar range. Six times higher effective transporter concentrations were measured with aminonaphthalene-1,3,6-trisulfonate than with 5(6)-carboxyfluorescein, demonstrating the importance of anion binding for transport and excluding nonspecific efflux. Negative surface potentials efficiently annihilate transport activity, while inside-negative membrane potentials slightly increase it. These trends demonstrate the functional importance of counterions to hinder the binding of hydrophilic counterions and to minimize the global positive charge of the transporter−counterion complexes. Strong, nonlinear increases in activity with polymer length reveal a significant polymer effect. Overall, the characteristics of PGONs do not match those of similar systems (for example, polyarginine) and hint toward an interesting mode of action, clearly different from nonspecific leakage caused by detergents. The activity of PGONs increases in the presence of amphiphilic anions such as pyrenebutyrate (EC50 = 70 μM), while several other amphiphilic anions tested were inactive. PGONs are efficiently inactivated by numerous hydrophilic anions including ATP (IC50 = 150 μM), ADP (IC50 = 460 μM), heparin (IC50 = 1.0 μM), phytate (IC50 = 0.4 μM), and CB hydrazide (IC50 = 26 μM). The compatibility of this broad responsiveness with multicomponent sensing in complex matrices is discussed and illustrated with lactate sensing in sour milk. The PGON lactate sensor operates together with lactate oxidase as a specific signal generator and CB hydrazide as an amplifier for covalent capture of the pyruvate product as CB hydrazone (IC50 = 1.5 μM).

Comparison of facially amphiphilic versus segregated monomers in the design of antibacterial copolymers.

A direct comparison of two strategies for designing antimicrobial polymers is presented. Previously, we published several reports on the use of facially amphiphilic (FA) monomers which led to polynorbornenes with excellent antimicrobial activities and selectivities. Our polymers obtained by copolymerization of structurally similar segregated monomers, in which cationic and non-polar moieties reside on separate repeat units, led to polymers with less pronounced activities. A wide range of polymer amphiphilicities was surveyed by pairing a cationic oxanorbornene with eleven different non-polar monomers and varying the comonomer feed ratios. Their properties were tested using antimicrobial assays and copolymers possessing intermediate hydrophobicities were the most active. Polymer-induced leakage of dye-filled liposomes and microscopy of polymer-treated bacteria support a membrane-based mode of action. From these results there appears to be profound differences in how a polymer made from FA monomers interacts with the phospholipid bilayer compared with copolymers from segregated monomers. We conclude that a well-defined spatial relationship of the whole polymer is crucial to obtain synthetic mimics of antimicrobial peptides (SMAMPs): charged and non-polar moieties need to be balanced locally, for example, at the monomer level, and not just globally. We advocate the use of FA monomers for better control of biological properties. It is expected that this principle will be usefully applied to other backbones such as the polyacrylates, polystyrenes, and non-natural polyamides.

Self‐Activation in De Novo Designed Mimics of Cell‐Penetrating Peptides

The unique ability of cell-penetrating peptides (CPPs), also known as protein transduction domains, to navigate across the nonpolar biological membrane has been under intense investigation. In vitro studies have shown that multiple mechanisms are available, with the precise details being dependent on the peptide and cell line studied. The several clearly demonstrated pathways include various forms of endocytosis, macropinocytosis, lipid-raft-dependent macropinocytosis, and protein-dependent translocation. In addition, an energy-independent pathway, or spontaneous translocation, has also been illustrated. 5] Perhaps the clearest example of an energy-independent pathway is the ability of CPPs, and their synthetic mimics, to cross model phospholipid bilayer vesicle membranes. General consensus in the literature suggests that hydrophobic counterions play an essential role in this transduction by complexation around the guanidinium-rich backbone, thus coating the highly cationic structure with lipophilic moieties. For example, an octamer of arginine in the presence of sodium laurate partitioned into octanol versus water with better than 95% efficiency. Separately, it was shown that the simple peptide nonaarginine ((Arg)9) does not in fact transverse membranes very effectively on its own. However, the presence of hydrophobic counterions “activates” this molecule, thus turning it into a potent transduction peptide. It was shown that n-alkyl chain surfactants were good “activators” and thus efficient at promoting the transport of oligoand polyarginines across biological membranes. 8] After the initial discovery that CPP-like behavior could be emulated in simple norbornene-based polymers, we wondered if the presence of covalently attached hydrophobic residues would increase their translocation activity. To evaluate this hypothesis, we designed and synthesized a series of norbornene-based guanidine-rich polymers, where the hydrophobic groups were introduced through a side chain rather than as counterions (Scheme 1). Remarkably, the guanidine polymers containing certain alkyl side chains exhibited significantly enhanced activity (by three orders of magnitude) without the need for any “counterion activator”. Monomers were prepared by either Mitsunobu coupling or nucleophilic substitution reactions (see the Supporting Information). Random copolymers G1–G12 with 50:50 mol% monomer distribution were targeted at two molecular weights (Mn) using ring-opening metathesis polymerization (ROMP; low Mn 2.9–3.9 kDa and high Mn 11.4– 13.6 kDa of the tert-butyloxycarbonyl (Boc)-protected polymers were obtained). Gel-permeation chromatography gave monomodal signals and narrow molecular-weight indices (1.05–1.15). The Boc-protected polymers were deprotected to obtain G1–G12, and their activities were studied in vesicle assays. Using the standard biophysical assay well-accepted in the CPP literature, the transport activities of G1–G12 were determined. Specifically, 5(6)-carboxyfluorescein (CF) was used as a fluorescent probe in egg yolk phosphatidylcholine large unilamellar vesicles (EYPC-LUVs). The activity of G1– G12 transporters increased with increasing polymer content at a constant vesicle concentration as detected by CF emission intensity, yielding plots of fluorescence intensity versus polymer concentration for the series G1–G12 (Supporting Information, Figure S1). Fitting the Hill equation (Y/ (c/EC50) ) to this data for each individual polymer revealed a nonlinear dependence of the fractional fluorescence intensity Yon the polymer concentration c. This analysis gave Ymax (maximal CF release relative to complete release by Triton X100), EC50 (effective polymer concentration needed to reach Ymax/2), and the Hill coefficient n (Supporting Information, Figure S2, Tables S1 and S2). For direct comparison, it is worth mentioning that the CPPs heptaarginine and polyarginine were inactive under these conditions; it is known that polyarginine needs counterions for activation. Figure 1 collects the EC50 values for this series of copolymers. Polymers with lower EC50 values are said to be Scheme 1. Guanidino copolymers G1–G12.

Interactions between Antimicrobial Polynorbornenes and Phospholipid Vesicles Monitored by Light Scattering and Microcalorimetry

Antimicrobial polynorbornenes composed of facially amphiphilic monomers have been previously reported to accurately emulate the antimicrobial activity of natural host-defense peptides (HDPs). The lethal mechanism of most HDPs involves binding to the membrane surface of bacteria leading to compromised phospholipid bilayers. In this paper, the interactions between biomimetic vesicle membranes and these cationic antimicrobial polynorbornenes are reported. Vesicle dye-leakage experiments were consistent with previous biological assays and corroborated a mode of action involving membrane disruption. Dynamic light scattering (DLS) showed that these antimicrobial polymers cause extensive aggregation of vesicles without complete bilayer disintegration as observed with surfactants that efficiently solubilize the membrane. Fluorescence microscopy on vesicles and bacterial cells also showed polymer-induced aggregation of both synthetic vesicles and bacterial cells. Isothermal titration calorimetry (ITC) afforded free energy of binding values (Delta G) and polymer to lipid binding ratios, plus revealed that the interaction is entropically favorable (Delta S>0, Delta H>0). It was observed that the strength of vesicle binding was similar between the active polymers while the binding stoichiometries were dramatically different.

Synthesis of a FTO Inhibitor with Anticonvulsant Activity

We describe the rationale for and the synthesis of a new class of compounds utilizing a modular approach that are designed to mimic ascorbic acid and to inhibit 2-oxoglutarate-dependent hydroxylases. Preliminary characterization of one of these compounds indicates in vivo anticonvulsant activity (6 Hz mouse model) at nontoxic doses, inhibition of the 2-oxoglutarate-dependent hydroxylase FTO, and expected increase in cellular N(6)-methyladenosine. This compound is also able to modulate various microRNA, an interesting result in light of the recent view that modulation of microRNAs may be useful for the treatment of CNS disease.