Design of Triazolium-Grafted Peptidomimetic Macrocycles with Facial Amphipathicity to Target Pathogenic Bacteria.

Access to 1,2,3-triazolium-grafted peptoid macrocycles was developed by macrocyclization and multivalent postmodification of linear peptoid oligomers carrying an alternance of benzylic and propargyl groups as side chains. X-ray analysis and NMR studies revealed a conformational preference for constrained hairpin-shaped structures leading to the facial amphipathic character of these macrocycles. A preliminary evaluation showed the antimicrobial activities of these new cationic amphipathic architectures.

Dimeric peptoids as antibacterial agents.

Building upon our previous study on peptoid-based antibacterials which showed good activity against Gram-positive bacteria only, herein we report the synthesis of 34 dimeric peptoid compounds and the investigation of their activity against Gram-positive and Gram-negative pathogens. The newly designed peptoids feature a di-hydrophobic moiety incorporating phenyl, bromo-phenyl, and naphthyl groups, combined with variable lengths of cationic units such as amino and guanidine groups. The study also underscores the pivotal interplay between hydrophobicity and cationicity in optimizing efficacy against specific bacteria. The bromophenyl dimeric guanidinium peptoid compound 10j showed excellent activity against S. aureus 38 and E. coli K12 with MIC of 0.8 µg mL-1 and 6.2 µg mL-1, respectively. Further investigation into the mechanism of action revealed that the antibacterial effect might be attributed to the disruption of bacterial cell membranes, as suggested by tethered bilayer lipid membranes (tBLMs) and cytoplasmic membrane permeability studies. Notably, these promising antibacterial agents exhibited negligible toxicity against mammalian red blood cells. Additionally, the study explored the potential of 12 active compounds to disrupt established biofilms of S. aureus 38. The most effective biofilm disruptors were ethyl and octyl-naphthyl guanidinium peptoids (10c and 10 k). These compounds 10c and 10 k disrupted the established biofilms of S. aureus 38 with 51 % at 4x MIC (MIC = 17.6 µg mL-1 and 11.2 µg mL-1) and 56 %-58 % at 8x MIC (MIC = 35.2 µg mL-1 and 22.4 µg mL-1) respectively. Overall, this research contributes insights into the design principles of cationic dimeric peptoids and their antibacterial activity, with implications for the development of new antibacterial compounds.

Bio-instructive materials on-demand - combinatorial chemistry of peptoids, foldamers, and beyond.

Combinatorial chemistry allows for the rapid synthesis of large compound libraries for high throughput screenings in biology, medicinal chemistry, or materials science. Especially compounds from a highly modular design are interesting for the proper investigation of structure-to-activity relationships. Permutations of building blocks result in many similar but unique compounds. The influence of certain structural features on the entire structure can then be monitored and serve as a starting point for the rational design of potent molecules for various applications. Peptoids, a highly diverse class of bioinspired oligomers, suit perfectly for combinatorial chemistry. Their straightforward synthesis on a solid support using repetitive reaction steps ensures easy handling and high throughput. Applying this modular approach, peptoids are readily accessible, and their interchangeable side-chains allow for various structures. Thus, peptoids can easily be tuned in their solubility, their spatial structure, and, consequently, their applicability in various fields of research. Since their discovery, peptoids have been applied as antimicrobial agents, artificial membranes, molecular transporters, and much more. Studying their three-dimensional structure, various foldamers with fascinating, unique properties were discovered. This non-comprehensive review will state the most interesting discoveries made over the past years and arouse curiosity about what may come.

Design and synthesis of a DNA-encoded combinatorial library of bicyclic peptoids.

Here we describe the design and synthesis of a DNA-encoded library of bicyclic peptoids. We show that our solid-phase strategy is facile and DNA-compatible, yielding a structurally diverse combinatorial library of bicyclic peptoids of various ring sizes. We also demonstrate that affinity-based screening of a DNA-encoded library of bicyclic peptoids enables to efficiently identify high-affinity ligands for a target protein. Given their highly constraint structures, as well as increased cell permeability and proteolytic stability relative to native peptides, bicyclic peptoids could be an excellent source of protein capture agents. As such, our DNA-encoded library of bicyclic peptoids will serve as versatile tools that facilitate the generation of potent ligands against many challenging targets, such as intracellular protein-protein interactions.

Macrocyclic Tetramers-Structural Investigation of Peptide-Peptoid Hybrids.

Outstanding affinity and specificity are the main characteristics of peptides, rendering them interesting compounds for basic and medicinal research. However, their biological applicability is limited due to fast proteolytic degradation. The use of mimetic peptoids overcomes this disadvantage, though they lack stereochemical information at the α-carbon. Hybrids composed of amino acids and peptoid monomers combine the unique properties of both parent classes. Rigidification of the backbone increases the affinity towards various targets. However, only little is known about the spatial structure of such constrained hybrids. The determination of the three-dimensional structure is a key step for the identification of new targets as well as the rational design of bioactive compounds. Herein, we report the synthesis and the structural elucidation of novel tetrameric macrocycles. Measurements were taken in solid and solution states with the help of X-ray scattering and NMR spectroscopy. The investigations made will help to find diverse applications for this new, promising compound class.

Peptide/ß-Peptoid Hybrids with Ultrashort PEG-Like Moieties: Effects on Hydrophobicity, Antibacterial Activity and Hemolytic Properties.

PEGylation of antimicrobial peptides as a shielding tool that increases stability toward proteolytic degradation typically leads to concomitant loss of activity, whereas incorporation of ultrashort PEG-like amino acids (sPEGs) remains essentially unexplored. Here, modification of a peptide/ß-peptoid hybrid with sPEGs was examined with respect to influence on hydrophobicity, antibacterial activity and effect on viability of mammalian cells for a set of 18 oligomers. Intriguingly, the degree of sPEG modification did not significantly affect hydrophobicity as measured by retention in reverse-phase HPLC. Antibacterial activity against both wild-type and drug-resistant strains of Escherichia coli and Acinetobacter baumannii (both Gram-negative pathogens) was retained or slightly improved (MICs in the range 2-16 µg/mL equal to 0.7-5.2 µM). All compounds in the series exhibited less than 10% hemolysis at 400 µg/mL. While the number of sPEG moieties appeared not to be clearly correlated with hemolytic activity, a trend toward slightly increased hemolytic activity was observed for analogues displaying the longest sPEGs. In contrast, within a subseries the viability of HepG2 liver cells was least affected by analogues displaying the longer sPEGs (with IC50 values of ~1280 µg/mL) as compared to most other analogues and the parent peptidomimetic (IC50 values in the range 330-800 µg/mL).

Design, synthesis and cytotoxic evaluation of peptoid analogs of an anticancer active triazolylpeptidyl penicillin.

Aim: Encouraged by the antitumor activity exhibited by triazolylpeptidyl penicillins, we decided to synthesize and evaluate a library of peptoid analogs. Results: The replacement of the dipeptide unit of the reference compound, TAP7f, was investigated. In addition, the effect of the triazole linking group on the biological activity of these new derivatives was evaluated, exchanging it with a glycine spacer. The cytotoxic effect of the library compounds was determined in the B16-F0 cell line and compared with the effects on normal murine mammary gland cells. Conclusion: Among the tested compounds, peptoid 4e exhibited the highest antiproliferative activity.

Formation of a tris(catecholato) iron(III) complex with a nature-inspired cyclic peptoid ligand.

Siderophore-mimicking macrocyclic peptoids were synthesized. Peptoid 3 with intramolecular hydrogen bonds showed an optimally arranged primary coordination sphere leading to a stable catecholate-iron complex. The tris(catecholato) structure of 3-Fe(iii) was determined with UV-vis, fluorescence, and EPR spectroscopies and DFT calculations. The iron binding affinity was comparable to that of deferoxamine, with enhanced stability upon air exposure.

Alternating Cationic-Hydrophobic Peptide/Peptoid Hybrids: Influence of Hydrophobicity on Antibacterial Activity and Cell Selectivity.

The influence of hydrophobicity on antibacterial activity versus the effect on the viability of mammalian cells for peptide/peptoid hybrids was examined for oligomers based on the cationic Lys-like peptoid residue combined with each of 28 hydrophobic amino acids in an alternating sequence. Their relative hydrophobicity was correlated to activity against both Gram-negative and Gram-positive species, human red blood cells, and HepG2 cells. This identified hydrophobic side chains that confer potent antibacterial activity (e. g., MICs of 2-8 µg/mL against E. coli) and low toxicity toward mammalian cells (<10 % hemolysis at 400 µg/mL and IC50 >800 µg/mL for HepG2 viability). Most peptidomimetics retained activity against drug-resistant strains. These findings corroborate the hypothesis that for related peptidomimetics two hydrophobicity thresholds may be identified: i) it should exceed a certain level in order to confer antibacterial activity, and ii) there is an upper limit, beyond which cell selectivity is lost. It is envisioned that once identified for a given subclass of peptide-like antibacterials such thresholds can guide further optimisation.

Mechanistic Studies of the Multiple Myeloma and Melanoma Cell-Selective Toxicity of the Rpn13-Binding Peptoid KDT-11.

We previously reported a peptoid ligand for the proteasomal ubiquitin receptor Rpn13 called KDT-11 and demonstrated that this compound is toxic to multiple myeloma cells, but not non-malignant cells. Here, we show that KDT-11 decreases the viability of a variety of cancer cell lines, especially melanomas and various blood cancers. The peptoid induces selective G1 cell-cycle arrest, resulting in eventual apoptosis. While KDT-11 does not antagonize any of the known protein-protein interactions involving Rpn13, the peptoid inhibits the ability of Rpn13 to stimulate the activity of an associated deubiquitylase Uch37/UCHL5 in vitro, suggesting a high level of Uch37 activity might be important for cancer cell proliferation. However, a variety of experiments in SK-MEL-5 melanoma cells suggest that KDT-11's cytotoxic effects are mediated by interactions with proteins other than Rpn13.

Mass spectrometry studies of the fragmentation patterns and mechanisms of protonated peptoids.

Peptoids belong to a class of sequence-controlled polymers comprising of N-alkylglycine. This study focuses on using tandem mass spectrometry techniques to characterize the fragmentation patterns of a set of singly and doubly protonated peptoids consisting of one basic residue placed at different positions. The singly protonated peptoids fragment by producing predominately high-abundant C-terminal ions called Y-ions and low-abundant N-terminal ions called B-ions. Computational studies suggest that the proton affinity (PA) of the C-terminal fragments is generally higher than that of the N-terminal fragments, and the PA of the former increases as the fragments are elongated. The B-ions are likely formed upon dissociating the proton-activated amide bonds via an oxazolone structure, and the Y-ions are produced subsequently by abstracting a proton from the newly formed B-ions, which is energetically favored. The doubly protonated peptoids prefer to fragment closest to either the N- or the C-terminus and produce corresponding B/Y-ion pairs. The basic residue seems to dictate the preferred fragmentation site, which may be the result of minimizing the repulsion between the two charges. Water and terminal neutral losses are a facile process accompanying the peptoid fragmentation in both charge states. The patterns appear to be highly influenced by the location of the basic residue.

Evaluation of peptoid mimics of short, lipophilic peptide antimicrobials.

INTRODUCTION: Antimicrobial peptides are proving to be promising lead compounds for therapeutics. The major disadvantage of antimicrobial peptides is their proteolytic instability in the body, with half-lives averaging less than an hour. Peptoids, or N-substituted glycines, have emerged as a promising field of peptidomimetics by retaining the beneficial properties of antimicrobial peptides while improving their stability. METHODS: This study evaluated peptoid derivatives of ultra-short lipophilic antimicrobial peptides, comparing their potency side-by-side with the most prevalent multidrug-resistant bacteria (ESKAPE) and yeast pathogens (Candida albicans and Cryptococcus neoformans). RESULTS: Both peptide and peptoid counterparts were most effective against Gram-positive bacteria with minimum inhibitory concentration (MIC) values as low as 1.6 and 6.3 µg/mL, respectively. In general, peptides retained better antimicrobial activity than their peptoid counterparts; however, certain peptoids proved to be more effective than peptides against Gram-negative bacteria. For example, peptoid MG10 displayed an MIC of 6.3 µg/mL against Pseudomonas aeruginosa compared with the peptide counterpart with an MIC of 100 µg/mL. All tested compounds were more potent against Cryptococcus neoformans compared with Candida albicans. Cytotoxicity analysis indicated that peptoids were generally slightly less toxic than their peptide counterparts. Additionally, trypsin rapidly degraded one of the evaluated peptides, while having no effect on comparable peptoids, demonstrating the proteolytic stability of peptoids. CONCLUSION: These results indicate that direct conversion of lipopeptides to lipopeptoids can result in compounds with comparable antimicrobial activity, favorable mammalian cell toxicity, and excellent proteolytic stability.

Diblock copolypeptoids: a review of phase separation, crystallization, self-assembly and biological applications.

Polypeptoids are biocompatible, synthetically accessible, chemically and enzymatically stable, chemically diverse, and structurally controllable. As a bioinspired and biomimetic material, it has attracted considerable attention due to its great potential in biological applications including drug and gene delivery, sensing, imaging, molecular recognition, and anti-cancer therapy. Diblock copolypeptoids have especially been of increasing interest in the materials chemistry community because of their capacity to microphase separate and self-assemble to form a variety of nanoarchitectures. This review will discuss recent studies on diblock copolypeptoids regarding their synthesis, microphase separation, crystallization, self-assembly, and biological applications.

Peptoid-based siderophore mimics as dinuclear Fe3+ chelators.

A practical synthesis of preorganized tripodal enterobactin/corynebactin-type ligands (consisting of a C3-symmetric macrocyclic peptoid core, three catecholamide coordinating units, and C2, C4, and C6 spacers) is reported. The formation of complexes with Fe3+ was investigated by spectrophotometric (UV-Vis) and spectrometric (ESI, negative ionization mode) methods and corroborated by theoretical (DFT) calculations. Preliminary studies revealed the intricate interplay between the conformational chirality of cyclic trimeric peptoids and metal coordination geometry of mononuclear species similar to that of natural catechol-based siderophores. Experimental results demonstrated the unexpected formation of unique dinuclear Fe3+ complexes.

DNA origami protection and molecular interfacing through engineered sequence-defined peptoids.

DNA nanotechnology has established approaches for designing programmable and precisely controlled nanoscale architectures through specific Watson-Crick base-pairing, molecular plasticity, and intermolecular connectivity. In particular, superior control over DNA origami structures could be beneficial for biomedical applications, including biosensing, in vivo imaging, and drug and gene delivery. However, protecting DNA origami structures in complex biological fluids while preserving their structural characteristics remains a major challenge for enabling these applications. Here, we developed a class of structurally well-defined peptoids to protect DNA origamis in ionic and bioactive conditions and systematically explored the effects of peptoid architecture and sequence dependency on DNA origami stability. The applicability of this approach for drug delivery, bioimaging, and cell targeting was also demonstrated. A series of peptoids (PE1-9) with two types of architectures, termed as "brush" and "block," were built from positively charged monomers and neutral oligo-ethyleneoxy monomers, where certain designs were found to greatly enhance the stability of DNA origami. Through experimental and molecular dynamics studies, we demonstrated the role of sequence-dependent electrostatic interactions of peptoids with the DNA backbone. We showed that octahedral DNA origamis coated with peptoid (PE2) can be used as carriers for anticancer drug and protein, where the peptoid modulated the rate of drug release and prolonged protein stability against proteolytic hydrolysis. Finally, we synthesized two alkyne-modified peptoids (PE8 and PE9), conjugated with fluorophore and antibody, to make stable DNA origamis with imaging and cell-targeting capabilities. Our results demonstrate an approach toward functional and physiologically stable DNA origami for biomedical applications.

Synthesis of Information-bearing Peptoids and their Sequence-directed Dynamic Covalent Self-assembly.

This protocol presents the use of Lewis acidic multi-role reagents to circumvent kinetic trapping observed during the self-assembly of information-encoded oligomeric strands mediated by paired dynamic covalent interactions in a manner mimicking the thermal cycling commonly employed for the self-assembly of complementary nucleic acid sequences. Primary amine monomers bearing aldehyde and amine pendant moieties are functionalized with orthogonal protecting groups for use as dynamic covalent reactant pairs. Using a modified automated peptide synthesizer, the primary amine monomers are encoded into oligo(peptoid) strands through solid-phase submonomer synthesis. Upon purification by high-performance liquid chromatography (HPLC) and characterization by electrospray ionization mass spectrometry (ESI-MS), sequence-specific oligomers are subjected to high-loading of a Lewis acidic rare-earth metal triflate which both deprotects the aldehyde moieties and affects the reactant pair equilibrium such that strands completely dissociate. Subsequently, a fraction of the Lewis acid is extracted, enabling annealing of complementary sequence-specific strands to form information-encoded molecular ladders characterized by matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS). The simple procedure outlined in this report circumvents kinetic traps commonly experienced in the field of dynamic covalent assembly and serves as a platform for the future design of robust, complex architectures.

Sequence-selective dynamic covalent assembly of information-bearing oligomers.

Relatively robust dynamic covalent interactions have been employed extensively to mediate molecular self-assembly reactions; however, these assembly processes often do not converge to a thermodynamic equilibrium, instead yielding mixtures of kinetically-trapped species. Here, we report a dynamic covalent self-assembly process that mitigates kinetic trapping such that multiple unique oligomers bearing covalently coreactive pendant groups are able to undergo simultaneous, sequence-selective hybridization with their complementary strands to afford biomimetic, in-registry molecular ladders with covalent rungs. Analogous to the thermal cycling commonly employed for nucleic acid melting and annealing, this is achieved by raising and lowering the concentration of a multi-role reagent to effect quantitative dissociation and subsequently catalyze covalent bond rearrangement, affording selective assembly of the oligomeric sequences. The hybridization specificity afforded by this process further enabled information encoded in oligomers to be retrieved through selective hybridization with complementary, mass-labeled sequences.

One-pot organocatalytic/multicomponent approach for the preparation of novel enantioenriched non-natural selenium-based peptoids and peptide-peptoid conjugates.

A combined organocatalytic and multicomponent synthetic approach was designed for the preparation of selenium-based peptoids and peptide-peptoid conjugates. This single-step synthetic protocol comprises the organocatalytic asymmetric insertion of phenylselenium in the aldehyde moiety followed by the Ugi four-component reaction which results in obtaining the desired compounds in good-to-moderate yields and with good-to-excellent levels of stereoselectivity.

Discovery of Stable and Selective Antibody Mimetics from Combinatorial Libraries of Polyvalent, Loop-Functionalized Peptoid Nanosheets.

The ability of antibodies to bind a wide variety of analytes with high specificity and high affinity makes them ideal candidates for therapeutic and diagnostic applications. However, the poor stability and high production cost of antibodies have prompted exploration of a variety of synthetic materials capable of specific molecular recognition. Unfortunately, it remains a fundamental challenge to create a chemically diverse population of protein-like, folded synthetic nanostructures with defined molecular conformations in water. Here we report the synthesis and screening of combinatorial libraries of sequence-defined peptoid polymers engineered to fold into ordered, supramolecular nanosheets displaying a high spatial density of diverse, conformationally constrained peptoid loops on their surface. These polyvalent, loop-functionalized nanosheets were screened using a homogeneous Förster resonance energy transfer (FRET) assay for binding to a variety of protein targets. Peptoid sequences were identified that bound to the heptameric protein, anthrax protective antigen, with high avidity and selectivity. These nanosheets were shown to be resistant to proteolytic degradation, and the binding was shown to be dependent on the loop display density. This work demonstrates that key aspects of antibody structure and function-the creation of multivalent, combinatorial chemical diversity within a well-defined folded structure-can be realized with completely synthetic materials. This approach enables the rapid discovery of biomimetic affinity reagents that combine the durability of synthetic materials with the specificity of biomolecular materials.

Synthesis and Application of an Activity-Based Peptide-Peptoid Hybrid Probe for the Immunoproteasome.

The immunoproteasome (iCP), a specific isoform of the proteasome's catalytic particle, is becoming an important protein complex of interest in various diseases. However, there is still much left to be learned about its activity in cells and how this can be altered by various endogenous conditions or with treatment with small molecules. Current strategies to investigate the iCP lack in their ability to be used in live, intact cells, limiting them to use in endpoint experiments. The iCP-selective probe presented here has been shown to be compatible with various live-cell assays, including monitoring iCP activity kinetically in a plate reader-based assay and observing single cells with confocal microscopy. A well-studied iCP-selective inhibitor, ONX-0914, has also been demonstrated to decrease the fluorescence signal of the iCP probe in both of these assays, showing its potential function in investigating small-molecule modulators of the iCP. © 2019 by John Wiley & Sons, Inc. Basic Protocol 1: Synthesis of an immunoproteasome-selective peptide-peptoid hybrid probe Basic Protocol 2: Expression of the immunoproteasome in A549 cells Basic Protocol 3: Using the immunoproteasome probe to monitor activity in live cells with a fluorescence plate reader Basic Protocol 4: Using the immunoproteasome probe to monitor activity in live cells with confocal microscopy.