Dual-functional ROMP-based betaines: effect of hydrophilicity and backbone structure on nonfouling properties

Foundational materials for nonfouling coatings were designed and synthesized from a series of novel dual-functional zwitterionic polymers, Poly[NRZI], which were easily obtained via ring-opening metathesis polymerization (ROMP) followed by a single step transformation of the cationic precursor, Poly[NR(+)], to the zwitterion, Poly[NRZI]. The resulting unique dual-functional structure contained the anion and the cation within the same repeat unit but on separate side chains, enabling the hydrophilicity of the system to be tuned at the repeat unit level. These dual-functional zwitterionic polymers were specifically designed to investigate the impact of structural changes, including the backbone, hydrophilicity, and charge, on the overall nonfouling properties. To evaluate the importance of backbone structure, and as a direct comparison to previously studied methacrylate-based betaines, norbornene-based carbo- and sulfobetaines (Poly[NCarboZI] and Poly[NSulfoZI]) as well as a methacrylate-based sulfobetaine (Poly[MASulfoZI]) were synthesized. These structures contain the anion-cation pairs on the same side chain. Nonfouling coatings were prepared from copolymers, composed of the zwitterionic/cationic precursor monomer and an ethoxysilane-containing monomer. The coatings were evaluated by using protein adsorption studies, which clearly indicated that the overall hydrophilicity has a major influence on the nonfouling character of the materials. The most hydrophilic coating, from the oligoethylene glycol (OEG)-containing dual-functional betaine, Poly[NOEGZI-co-NSi], showed the best resistance to nonspecific protein adsorption (Γ(FIB) = 0.039 ng/mm(2)). Both norbornene-based polymers systems, Poly[NSulfoZI] and Poly[NCarboZI], were more hydrophilic and thus more resistant to protein adsorption than the methacrylate-based Poly[MASulfoZI]. Comparing the protein resistance of the dual-functional zwitterionic coatings, Poly[NRZI-co-NSi], to that of their cationic counterparts, Poly[NR(+)-co-NSi], revealed the importance of screening electrostatic interactions. The adsorption of negatively charged proteins on zwitterionic coatings was significantly less, despite the fact that both coatings had similar wetting properties. These results demonstrate that the unique, tunable dual-functional zwitterionic polymers reported here can be used to make coatings that are highly efficient at resisting protein adsorption.     

Synthetic Mimics of Antimicrobial Peptides—A Versatile Ring‐Opening Metathesis Polymerization Based Platform for the Synthesis of Selective Antibacterial and Cell‐Penetrating Polymers

Natural macromolecules exhibit an extensive arsenal of properties, many of which have proven difficult to recapitulate in simpler synthetic systems. Over the last couple of years, foldamers have emerged as one important step toward increased functionality in synthetic systems. While the great majority of work in this area has focused on folded structures, hence the name, more recent progress has centered on polymers that mimic protein function. These efforts have resulted in the design of relatively simple macromolecules; one example are the synthetic mimics of antimicrobial peptides (SMAMPs) that capture the central physicochemical features of their natural archetypes irrespective of the specific folded form. Here we present our recent efforts to create polymers which display biological activity similar to natural proteins, including antimicrobial and cell‐penetrating peptides.     

“Doubly Selective” Antimicrobial Polymers: How Do They Differentiate between Bacteria?

We have investigated how doubly selective synthetic mimics of antimicrobial peptides (SMAMPs), which can differentiate not only between bacteria and mammalian cells, but also between Gram‐negative and Gram‐positive bacteria, make the latter distinction. By dye‐leakage experiments on model vesicles and complementary experiments on bacteria, we were able to relate the Gram selectivity to structural differences of these bacteria types. We showed that the double membrane of E. coli rather than the difference in lipid composition between E. coli and S. aureus was responsible for Gram selectivity. The molecular‐weight‐dependent antimicrobial activity of the SMAMPs was shown to be a sieving effect: while the 3000 g mol^?1 SMAMP was able to penetrate the peptidoglycan layer of the Gram‐positive S. aureus bacteria, the 50000 g mol^?1 SMAMP got stuck and consequently did not have antimicrobial activity.     

Antimicrobial Polymers Prepared by Ring‐Opening Metathesis Polymerization: Manipulating Antimicrobial Properties by Organic Counterion and Charge Density Variation

The synthesis and characterization of a series of poly(oxanorbornene)‐based synthetic mimics of antimicrobial peptides (SMAMPs) is presented. In the first part, the effect of different organic counterions on the antimicrobial properties of the SMAMPs was investigated. Unexpectedly, adding hydrophobicity by complete anion exchange did not increase the SMAMPs’ antimicrobial activity. It was found by dye‐leakage studies that this was due to the loss of membrane activity of these polymers caused by the formation of tight ion pairs between the organic counterions and the polymer backbone. In the second part, the effect of molecular charge density on the biological properties of a SMAMP was investigated. The results suggest that, above a certain charge threshold, neither minimum inhibitory concentration (MIC₉₀) nor hemolytic activity (HC₅₀) is greatly affected by adding more cationic groups to the molecule. A SMAMP with an MIC₉₀ of 4 μg mL^?1 against Staphylococcus aureus and a selectivity (=HC₅₀/MIC₉₀) of 650 was discovered, the most selective SMAMP to date.     

Solid-phase synthesis of a combinatorial array of 1,3-bis(acylamino)-2-butanones, inhibitors of the cysteine proteases cathepsins K and L

To more rapidly prepare members of the 1,3-bis(acylamino)-2-butanone class of cysteine protease inhibitors, a solid-phase synthesis was developed. 1-Azido-3-amino-2,2-dimethoxybutane (4), which has the two amino groups differentiated and the ketone protected as a a ketal, served as a surrogate for the 1,3-diamino-2-butanone core. Amine (4) was coupled to the BAL-resin-linked carboxylic acids derived from alpha-amino acid esters. Evaluation of a small combinatorial array by measuring inhibition constants (Ki,appS) against cathepsins K, L, and B provided some structure-activity relationship trends with respect to selectivity and potency. Novel, potent inhibitors of cathepsins K and L were identified.     

Theoretical study of helix formation in substituted phenylene ethynylene oligomers

Theoretical investigations of the relative stabilities of helical vs extended forms of phenylene ethynylene oligomers established that MMFF molecular mechanics was more useful than AM1 or DFT for calculating helical structures and for estimating relative energies. At the level of MMFF, theory predicts that for o- or m-oligophenylene ethynylenes, helix formation is enthalpically favored for ester and ether-substituted oligomers. In contrast to simple electron-demand predictions, we predict that the position of substituents can make a substantial difference in the tendency to form helices.     

Controlling the conformation of arylamides: computational studies of intramolecular hydrogen bonds between amides and ethers or thioethers

The role of an ortho-alkylthioether group in controlling the conformation around the ring-N bonds of meta-connected arylamide oligomers is studied. Density functional theory (DFT) geometries of model compounds, including acetanilide, an ether acetanilide, and a thioether acetanilide, and their corresponding diamides, show that for either monoamide or diamide the alkyl side chain of the thioether should be perpendicular to the aryl plane, whereas for the ether monoamide, the alkyl side chain is in the aryl plane. DFT ring-N torsional potentials and constrained geometries of the model compounds demonstrate that carbonyl-S repulsion leads to a high torsional barrier and that intramolecular N-H...S and C-H...O hydrogen bonds and ring-amide conjugation lead to N-H having a preferred orientation in the benzene plane pointing towards S. The N-H bond lengthens and the ortho-ring C-H bond shortens in a regular pattern in the approach to the preferred orientation. Calculated IR frequencies for the N-H stretch show a clear red shift between model compounds without and with the thioether side chain.     

Influence of lipid composition on membrane activity of antimicrobial phenylene ethynylene oligomers

Host defense peptides (HDPs), part of the innate immune system, selectively target the membranes of bacterial cells over that of host cells. As a result, their antimicrobial properties have been under intense study. Their selectivity strongly depends on the chemical and mostly structural properties of the lipids that make up different cell membranes. The ability to synthesize HDP mimics has recently been demonstrated. To better understand how these HDP mimics interact with bilayer membranes, three homologous antimicrobial oligomers (AMOs) 1-3 with an m-phenylene ethynylene backbone and alkyl amine side chains were studied. Among them, AMO 1 is nonactive, AMO 2 is specifically active, and AMO 3 is nonspecifically active against bacteria over human red blood cells, a standard model for mammalian cells. The interactions of these three AMOs with liposomes having different lipid compositions are characterized in detail using a fluorescent dye leakage assay. AMO 2 and AMO 3 caused more leakage than AMO 1 from bacteria membrane mimic liposomes composed of PE/PG lipids. The use of E. coli lipid vesicles gave the same results. Further changes of the lipid compositions revealed that AMO 2 has selectively higher affinity toward PE/PG and E. coli lipids than PC, PC/PG or PC/PS lipids, the major components of mammalian cell membranes. In contrast, AMO 3 is devoid of this lipid selectivity and interacts with all liposomes with equal ease; AMO 1 remains inactive. These observations suggest that lipid type and structure are more important in determining membrane selectivity than lipid headgroup charges for this series of HDP mimics.